Sap and peptidomimetics for treatment of eye disease

ABSTRACT

Self-assembling peptides or self-assembling peptidomimetics (“SAP”) can treat or alleviate disease, disorder, injury or one or more symptoms of diseases or disorders of the eye, including ocular inflammation, dry eye, corneal erosion, retinal detachment, and other problems where the barrier formed by the SAP provides protection and aids healing. SAP topical or injectable compositions of SAP for local administration to the eye include SAP in an amount and concentration effective to provide an SAP structure on or within the eye or a compartment or structure thereof. The SAP can be assembled prior to or after the composition is administration. SAP can also be used as coatings for contact lens, intraocular lens, and wound healing devices, to enhance healing and decrease inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Ser. No.62/647,184 filed Mar. 23, 2018, and which is incorporated by referencein its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Mar. 15, 2019 as a text file named“CNS_109_ST25.txt,” created on Mar. 15, 2019, and having a size of114,126 bytes is hereby incorporated by reference pursuant to 37 C.F.R.§ 1.52(e)(5).

FIELD OF THE INVENTION

This invention is in the field of therapeutic reagents, particularlycompositions of SAPs, which are useful for the treatment, prevention oralleviation of the symptoms of diseases, disorders, injuries and relatedsymptoms that affect the eye.

BACKGROUND OF THE INVENTION

The outer covering of the eyeball consists of a relatively tough, whitelayer called the sclera (or white of the eye). Near the front of theeye, in the area protected by the eyelids, the sclera is covered by athin, transparent membrane (conjunctiva), which runs to the edge of thecornea. The conjunctiva also covers the moist back surface of theeyelids and eyeballs.

Light enters the eye through the cornea, the clear, curved layer infront of the iris and pupil. The cornea serves as a protective coveringfor the front of the eye and also helps focus light on the retina at theback of the eye. After passing through the cornea, light travels throughthe pupil (the black dot in the middle of the eye). The iris, thecircular, colored area of the eye that surrounds the pupil, controls theamount of light that enters the eye. The iris allows more light into theeye, (enlarging or dilating the pupil, when the environment is dark andallows less light into the eye, (shrinking or constricting the pupil,when the environment is bright. The size of the pupil is controlled bythe action of the pupillary sphincter muscle and dilator muscle.

Behind the iris sits the lens. By changing its shape, the lens focuseslight onto the retina. Through the action of small muscles called theciliary muscles, the lens becomes thicker to focus on nearby objects andthinner to focus on distant objects. The retina contains the cells thatsense light (photoreceptors) and the blood vessels that nourish them.The most sensitive part of the retina is a small area called the macula,which has millions of tightly packed photoreceptors (the type calledcones). The high density of cones in the macula makes the visual imagedetailed, just as a high-resolution digital camera has more megapixels.Each photoreceptor is linked to a nerve fiber. The nerve fibers from thephotoreceptors are bundled together to form the optic nerve. The opticdisk, the first part of the optic nerve, is at the back of the eye. Thephotoreceptors in the retina convert the image into electrical signals,which are carried to the brain by the optic nerve. There are two maintypes of photoreceptors: cones and rods. Cones are responsible forsharp, detailed central vision and color vision and are clustered mainlyin the macula. Rods are responsible for night and peripheral vision.Rods are more numerous than cones and much more sensitive to light, butthey do not register color or contribute to detailed central vision asthe cones do. Rods are grouped mainly in the peripheral areas of theretina.

The eyeball is divided into two sections, each of which is filled withfluid. The pressure generated by these fluids fills out the eyeball andhelps maintain its shape. The front section (anterior segment) extendsfrom the inside of the cornea to the front surface of the lens and isfilled with aqueous humor, which nourishes the internal structures. Theanterior segment is divided into two chambers. The anterior chamberextends from the cornea to the iris. The posterior) chamber extends fromthe iris to the lens. Normally, the aqueous humor is produced in theposterior chamber, flows slowly through the pupil into the anteriorchamber, and then drains out of the eyeball through outflow channelslocated where the iris meets the cornea. The posterior segment extendsfrom the back surface of the lens to the retina. It contains a jellylikefluid called the vitreous humor.

The cornea is the dome-shaped, transparent outer surface of the front ofthe eye that exists directly beneath the pre-corneal tear film. The tearfilm is a thin layer of tears that covers the exposed area of the globeand contains an outer oily layer, a middle watery layer, and an innermucus layer. Light passes to the lens through the cornea, which providesapproximately two-thirds of the eye's total optical power. Theprotective, outermost layer of the cornea contains regenerativeepithelium that acts as a water permeable physical barrier.

Diseases, disorders, injuries of the eye, including the ocular surface,cornea, and other structures, can negatively impact eyesight and qualityof life and are often inadequately treated due to the need for moreeffective treatment options. Patients can experience a range of problemsand sequelae, including loss of visual acuity, blurry vision,photophobia, pain, discomfort, redness, itching, and blindness due tothe cornea, retina, and other structures. Chronic eye diseases anddisorders represent a significant healthcare burden. For example, theannual cost of managing DED in the United States has been estimated tobe 3.8 billion USD (Yu, et al., Cornea, 30(4): pp. 379-87 (2011);Waduthantril, et al., PLoS ONE, Vol 7(6), e37711 (2012)).

Dry Eye Disease (DED), also known as keratoconjunctivitis sicca (Lemp,et al., Report of the International Dry Eye WorkShop (DEWS) Ocul Surf 5:65-204 (2007)), is a multifactorial disease of the tears and ocularsurface that may cause discomfort, visual disturbance, tear filminstability and damage to the ocular surface. It is typicallyaccompanied by increased osmolarity of the tear film and inflammation atthe ocular surface. Treatment, which typically includes frequentapplications per day of artificial tears and non-prescription eye dropsto mitigate irritation and lubricate the eyes, usually provides onlyminimal and limited relief without modifying the disease course.

Recurrent corneal erosion syndrome (RCES) is a common clinical disordercharacterized by a disturbance at the level of the corneal epithelialbasement membrane resulting in defective adhesions and recurrentbreakdown of the epithelium. It may arise spontaneously or from anteriorbasement membrane dystrophy (e.g., Cogan dystrophy or map dotfingerprint dystrophy) or from other problems, such as adhesions betweenthe palpebral conjunctiva of the eyelids and the corneal epithelium.Management of RCES is usually aimed at regenerating or repairing theepithelial basement membrane to restore the adhesion between theepithelium and the anterior stroma. However, current limited options foreffectively controlling the development and progression of RCEStypically include application of lubricating ointment to prevent surfaceaggravation and antimicrobials to prevent and reduce infection of thedamaged mucosal tissue. Treatment can be prolonged and arduous, oftenleading to poor patient compliance poor therapeutic outcomes.

Although often unsuccessful, RCES treatment may include punctalocclusion, in which a plug is inserted into the tear duct, or directapplication of a bandage soft contact lens. Patients with refractoryRCES may undergo surgical interventions, such as Anterior StromalMicropuncture (ASM), phototherapeutic keratectomy and debridement of thecorneal epithelium, however, the attendant risks include scarring,glare, and blurred vision, and they often fail.

In many instances, eye disease prevalence and severity are affected byunderlying medical conditions. For instance, patients with diabetes aretwice as likely to suffer symptoms of DED and/or require frequent use ofartificial tear products. Elevated blood glucose levels can increasetear osmolarity and impair lacrimal gland tear secretion, resulting inDED. Also, increased glucose in the meibomian glands may disrupt thenormal flow of oil and lead to increased evaporation of tears andincreased the risk of microbial overgrowth.

Hyperglycemia also adversely affects adherence of corneal epithelialcells to underlying tissue, which can result in chronic sloughing of thecorneal surface characteristic of RCES and increased risk of infection.Current treatment regimens involving contact lenses and cornealrefractive surgery are intended for patients who maintain good glycemiccontrol (Working Together to Manage Diabetes: A guide for pharmacy,podiatry, Optometry, and dentistry, What Eye Care Professionals WouldLike Team Members to Know About Eye Health and Diabetes, pp. 59-67,2016).

In addition, prolonged periods of hyperglycemia can lead to retractionof neuronal processes in the cornea and corneal neuropathy (Yagihashi,et al., Journal of Diabetes Investigation, V.2 (1), pp. 18-32 (2011)),which in turn can delay intervention, diagnosis and treatment due toreduced sensitivity at the ocular surface, further exacerbating the eyediseases and their effects.

Treatment options for diseases, disorders, injuries and related symptomsof the corneal surface, such as DED and RCES, may also includeprescription eye drops containing immune-suppressants, such as topicalpreparations of cyclosporine (CsA; RESTASIS®), or corticosteroids (Lemp,Am J Manag Care.; 14(3 Suppl):S88-101(2008)). However, corticosteroidsare not ideal for long-term use due to possible side effects, and thistreatment regimen is often contraindicated in patients.

There remains a need for therapies that can be used to treat thesymptoms of eye disease, such as DED and RCES, while providing a barrierto protect the corneal epithelium from abrasion, contaminants, andinfective agents that contribute to disease progression and severity.

It is therefore an object of the present invention to providecompositions for treatment and prevention of one or more diseases,disorders, injuries and related symptoms that affect the eye.

It is also an object of the present invention to provide methods andcompositions for alleviating the symptoms of chronic DED.

It is also an object of the present invention to provide methods andcompositions for alleviating the symptoms of corneal erosions and RCES.

It is also an object of the present invention to provide methods andcompositions for reducing and preventing infection and enhancing repairof the diseased or damaged cornea.

It is still a further object of the present invention to provide methodsand compositions for preventing and treating diseases, disorders,injuries and related symptoms of the interior compartments of the eye.

SUMMARY OF THE INVENTION

Compositions of SAP and/or self-assembling peptidomimetics (referred toherein as “SAP”, unless designated otherwise), and methods of usethereof to control or treat diseases, disorders, injuries and relatedsymptoms that affect the eye by application directly onto or into theeye, are described. The SAP may be assembled prior to application, or beapplied as non-assembled precursor peptides and/or peptidomimetics,which assemble during or following application. Assembly can beinitiated upon contact with bodily fluids (e.g., tears at the surface ofthe eye) or an ionic solution.

In some embodiments, the SAP have a sequence of amino acid residuesconforming to one or more of the following formulas:

((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n);  (I)

((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n);  (II)

((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n);  (III) and

((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n),  (IV)

where each Xaa^(neu) represents an amino acid residue having a neutralcharge, Xaa⁺ represents an amino acid residue having a positive charge,Xaa⁻ represents an amino acid residue having a negative charge, x and yare integers having a value of 1, 2, 3, or 4, independently, and n is aninteger having a value of 1-5.

In certain embodiments, up to 100% of the SAP in the composition are ofthe same size and have the same amino acid sequence, for example, 75% ormore, such as 80%, 85%, 90% or 95%, or 99% of the SAP are of the samesize and have the same amino acid sequence. In other embodiments,compositions include two or more different SAP having different sizesand/or sequences. The compositions can also include polymers and can bepartly biodegradable, fully biodegradable, or non-biodegradable.Compositions can optionally include a separate scaffold or supportmaterial.

The SAP may contain a tissue specific targeting or binding sequence orligand. This may be mucoadhesive or targeted to specific cell types,such as epithelial or endothelial cells.

Compositions including one or more SAP can control, prevent or treatdiseases, disorders, injuries and related symptoms that affect the eye,provide an optically clear barrier to infection in and contamination ofthe cornea and other eye structures, and prevent or mitigateinflammation of the corneal stroma, retina and other eye structures.

Compositions including one or more SAP may contain one or moretherapeutic, prophylactic, diagnostic agents, and/or cells. Examplesinclude anti-inflammatories, anesthetics, antimicrobials, angiogenesisinhibitors, immunosuppressants, chemotherapeutics, ocularanti-hypertensive agents, and combinations thereof.

In preferred embodiments, the SAP compositions are formulated fortopical administration to the eye or for injection. In some embodiments,the SAP penetrates and transports the bound agent across the cornealsurface. The compositions are suitable for administration into one ormore of the internal compartments of the eye or onto the surface of theeye by, for example, instillation or injection. Systems for the topicalor intratissue application of compositions including SAP into or ontothe surface of the eye have been developed. Examples includeeye-dropper, sprays, and syringes. The amount and concentration of theSAP applied to the eye is typically sufficient to form an SAP structureat the surface of the eye, which may act as a barrier to the passage offluid. The SAP structure is optically clear, which can act as a barrierto prevent contamination and/or infection of the eye. Contact lenses,lacrimal inserts, and lens replacements can be coated on one or moresurface with SAP and optionally one or more therapeutic, prophylactic ordiagnostic agents. In some embodiments, the contact lenses include abacking material or support scaffold, which may be non-peptide.

Methods of making compositions that contain SAP for administration tothe eye can include injection molding, stamping, templating onto asurface having a desired shape, coating of a solid substrate,electro-spinning, or combinations of these. The SAP can be assembled bycontacting the composition with a solution of cations. Self-assembly ofthe peptides can occur at the time of manufacture of the composition, orimmediately prior to, during or after application of the composition tothe eye.

In some embodiments, the SAP are applied as eye drops to the eye of asubject to treat or prevent one or more diseases of the eye such asdiabetic retinopathy, retinitis pigmentosa, uveitis, inflammatory eyediseases, autoimmune eye diseases, dry eye syndrome (DED), Reiter'ssyndrome, psoriasis, rheumatoid arthritis, Sjogren's Syndrome, recurrentcorneal erosion syndrome (RCES), optic neuritis, and sarcoidosis. Incertain embodiments, the methods prevent the development or progressionof DED or RCES. The patient may suffer from a primary, secondary, oracquired metabolic disorder, such as diabetes. In some embodiments, theadministration of compositions of SAP enhances the healing of one ormore damaged or diseased structures at the surface of the eye. Forexample, in some embodiments, the methods decrease the amount of timerequired for the various layers of the cornea to heal by at least about10%, at least about 30%, or at least about 50%, relative to an untreatedcontrol.

In some embodiments, methods for the formation of an SAP structure atthe surface of the eye include the successive applications of one ormore SAP to the eye, to produce a multi-layered structure at the ocularsurface. When multiple layers of SAP structures are provided, each layercan include a specific peptide sequence or mixture of peptide sequences,having the same or different properties, as desired. For example, insome embodiments, two or more different SAP layers are consecutivelydeposited onto the surface of the eye. In some embodiments, eachapplication forms an SAP structure having different properties relativeto the other one or more layers. Exemplary properties include thickness,flexibility, the ability to bind or adhere to tissue, presence orhydrophobic or hydrophilic moieties, and the presence of one or moreadditional agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic depicting a cross-section view of the human eye.FIG. 1B is a schematic depicting a cross-section view of the layers ofthe tear film at the corneal surface, including the external lipid,middle aqueous, and inner mucus layers as well as the epithelial layerand corneal stroma. FIG. 1C is a schematic depicting a cross-sectionview of the cornea and tear ducts.

FIG. 2 is a histogram depicting the JEB score (−20 to 200) for each ofNormal, Saline Day 3, contralateral eye LPS Day 3, 1 Day LPS, 3 Day LPS,0.5% RADA+LPS Day 1, 0.5% RADA+LPS Day 3, and 0.5% RADA+LPS Day 7,respectively.

FIG. 3 is a histogram depicting the total retinal area weight-averageddensity (−200 to 1,200) for each of Normal, Saline Day 3, contralateraleye LPS Day 3, 1 Day LPS, 3 Day LPS, 0.5% RADA+LPS Day 1, 0.5% RADA+LPSDay 3, and 0.5% RADA+LPS Day 7, respectively.

FIG. 4 is a graph showing quantification of activated retinal microglialcells as an indicator of inflammation. The data is presented as pixelintensity and is normalized to remove variation across sections. Thepixel intensity from each of the layers of the eye across the varioustested conditions are presented. LPS alone (Ctrl L), or LPS incombination with (RADA)₂ at a concentration of 0.1% (S1-1), 1.0%(S1-10), and 10.0% (S1-100). S+A+R are the retina, sclera, and pigmentepithelium.

FIG. 5 is a graph showing quantification of inflammation. The y-axisindicates pixel density normalized to area as an indicator of activatedretinal microglial cells. S2: H₂N(EARA)COOH (SEQ ID NO: 92); S3:H₂N(RARA)CONH₂ (SEQ ID NO: 413); S5: H₂N(EARA)₂CONH₂ (SEQ ID NO: 414).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “about” is intended to describe values either above or belowthe stated value in a range of approximately +/−10%; in otherembodiments the values may range in value either above or below thestated value in a range of approximately +/−5%; in other embodiments thevalues may range in value either above or below the stated value in arange of approximately +/−2%; in other embodiments the values may rangein value either above or below the stated value in a range ofapproximately +/−1%. The preceding ranges are intended to be made clearby context, and no further limitation is implied.

“Biocompatible” refers to compatibility with living tissue or a livingsystem by not being toxic, injurious, or physiologically reactive andnot causing immunological rejection. Biocompatible materials, along withany metabolites or degradation products thereof, are generally non-toxicand do not cause significant adverse effects to the recipient.Biocompatible materials generally do not elicit a significant orproblematic inflammatory or immune response when administered.

“Biodegradable” generally refers to a material that under physiologicconditions degrades or erodes to smaller units or chemical species thatare capable of being metabolized, eliminated, or excreted by thesubject. The degradation time is a function of composition andmorphology and can last, for instance, hours, weeks or months.Degradation can include disassembly of SAP structures. Therefore, insome embodiments, degradation can include disassembly of SAP structures.

“Complementary” means having the capability of forming ionic or hydrogenbonding interactions between hydrophilic residues in adjacent peptidesin a structure. Hydrophilic residues in a peptide contain eitherhydrogen bonds or ionically pairs with a hydrophilic residue on anadjacent peptide, or is exposed to solvent. In most cases the peptidesassemble hydrophobic to hydrophobic and hydrophilic to hydrophilic,although hydrophobic to hydrophilic can occur. The structure of themolecule will change during assembly over time. Alignment can and doeschange during packing. In the case of an SAP such as RADA (SEQ ID NO:57), the hydrophobic face will assemble while the hydrophilic face willassemble in aqueous solvent; in the case of an oil solvent, the peptidewill assemble hydrophilic—hydrophilic, with the hydrophobic faceoriented into the oil. Pairing may also involve van der Waals forces.

“Effective amount refers to the amount necessary to elicit a desiredresponse, which may vary depending on such factors as the desiredoutcome, the agent being delivered, the nature of the target site, thenature of the conditions under which the agent is administered, etc. Forexample, the effective amount of a composition for treatment of adisease or disorder may be an amount sufficient to promote recovery to agreater extent than would occur in the absence of the composition.

“Preventing” refers to reducing the risk that a condition, state,disease, or symptom, or the manifestation or worsening thereof, willoccur.

The terms “treat”, “treatment” and “treating” refer to the reduction oramelioration of the progression, severity and/or duration of one or moresymptoms of an injury, disease or disorder, delay of the onset of adisease or disorder, or the amelioration of one or more consequences,indications or symptoms (preferably, one or more discernible symptoms)of an injury, disease or disorder, resulting from the administration ofone or more therapies (e.g., one or more therapeutic agents such as acompound as described).

“Increase,” “enhance,” “stimulate,” “induce” and/or like terms generallyrefer to the act of directly or indirectly improving or increasing afunction or behavior relative to its natural, expected, or average orrelative to current conditions.

The term “self-assembling” refers to the spontaneous or induced assemblyof molecules into defined, stable, non-covalently bonded structures thatare held together by intermolecular and/or intramolecular forces.

The term “topical administration” means non-invasive administration to atissue, organ, or orifice. Topical administrations can be administeredlocally and can provide a local effect in the region of applicationwhile limiting systemic exposure. Topical formulations can providesystemic effect via adsorption into the blood stream of the individual.Topical administration can include, but is not limited to, ophthalmic,cutaneous, transdermal, intravesicular, or mucosal (rectal, buccal,intranasal, or intravaginal, and rectal) administration.

“Small Molecule” refers to a molecule having a relatively low molecularweight, such as less than about 1000 or 1,500 g/mol. Typically, smallmolecules are not peptides or nucleic acids.

The term “carrier” or “excipient” refers to an organic or inorganic,natural or synthetic inactive ingredient in a formulation, with whichone or more additional ingredients are combined. Typically a carrier oran excipient is an inert substance added to a pharmaceutical compositionto further facilitate its administration, does not interfere with itsactivity or properties, and/or does not cause significant irritation tothe recipient.

II. Compositions

A. SAPs

Compositions for the treatment and prevention of eye disease includeSAP, amino acid residues or peptidomimetics that are capable ofself-assembly, or combinations thereof. In some embodiments, SAPcompositions include mixtures of self-assembling peptides andself-assembling peptidomimetics. In other embodiments, SAP includecombinations of standard amino acids and non-standard amino acids.

1. SAP

The term “peptide” includes “polypeptide,” “oligopeptide,” and“protein,” and refers to a chain of at least two α-amino acid residueslinked together by covalent bonds (peptide bonds). “Peptide” may referto an individual peptide or to a collection of peptides having the sameor different sequences, any of which may contain naturally occurringα-amino acid residues, non-naturally occurring α-amino acid residues,and combinations thereof. In particular, the D-enantiomer (“D-α-aminoacid”) of residues may be used. When D-α-amino acid residues (Xaa) areincluded within a sequence, they are annotated as “Xaa^(D)”. α-Aminoacid analogs are also known in the art and may be employed.Non-naturally occurring amino acids are not found or have not been foundin nature, but they can by synthesized and incorporated into a peptidechain. Suitable non-naturally occurring amino acids include, but are notlimited to, D-alloisoleucine(2R,3S)-2-amino-3-methylpentanoic acid,L-cyclopentyl glycine (S)-2-amino-2-cyclopentyl acetic acid.

Peptides can be represented as amino acid residue sequences. Thosesequences are written left to right in the direction from the amino(“N—”) to the carboxyl (“—C”) terminus. In accordance with standardnomenclature, amino acid residue sequences are denominated by either athree letter or a single letter code as indicated as follows: Alanine(Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp,D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E),Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu,L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F),Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp,W), Tyrosine (Tyr, Y), and Valine (Val, V). A “variant” of a peptiderefers to a polypeptide that differs from a reference polypeptide butretains essential properties. A variant and reference polypeptide maydiffer in amino acid sequence by one or more modifications (e.g.,substitutions, additions, and/or deletions of one or more residues)relative to the reference peptide.

Modifications and changes (e.g., “conservative amino acidsubstitutions”) can be made in the structure of the polypeptides withoutsubstantially affecting the self-assembly characteristics of thepolypeptide. For example, certain amino acids can be substituted forother amino acids in a sequence without appreciable variation inactivity. In making such changes, the hydropathic index of amino acidscan be considered. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar functionalactivity. It is known in the art that an amino acid can be substitutedby another amino acid having a similar hydropathic index and stillobtain a functionally equivalent polypeptide.

Substitution of like amino acids can also be made on the basis ofcharge. In certain embodiments, the substitution of amino acids havingan equivalent charge under physiological conditions can be made in thestructure of the polypeptides of the disclosure without substantiallyaffecting the self-assembly characteristics of the polypeptide. Chargestates negative (“−ve”), positive (“+ve”), and non-charged or neutral(“neu”) can be assigned to amino acid residues under physiologicalconditions as follows: aspartate (−ve); glutamate (−ve); arginine (+ve);lysine (+ve); histidine (neu or +ve); serine (neu); asparagine (neu);glutamine (neu); glycine (neu); proline (neu); threonine (neu); alanine(neu); cysteine (neu); methionine (neu); valine (neu); leucine (neu);isoleucine (neu); tyrosine (neu); phenylalanine (neu); tryptophan (neu).

Useful peptides can vary in length as long as they retain the ability toself-assemble to an extent useful for one or more of the purposes. Thenumber of amino acid residues in the peptide may range from as few asfour α-amino acid residues to as many as 100 residues. Typically,peptides which self-assemble have from about 6 to about 64 residues,more preferably from about 8 to about 36 residues, most preferably fromabout 8 to about 16 residues. In preferred embodiments, the peptide hasfrom about 8 to about 12 residues or about 12 to about 16 residues, orabout 16 to about 20 residues. In yet another embodiment, the peptidehas from about 16 to about 24 residues, or from about 16 to about 28residues, or from about 16 to about 32 residues.

One or more amino acid residues in an SAP can be altered or derivatizedby the addition of one or more chemical entities including, but notlimited to, acyl groups, carbohydrate groups, carbohydrate chains,phosphate groups, farnesyl groups, isofarnesyl groups, fatty acidgroups, or a linker which allows for conjugation or functionalization ofthe peptide. For example, either or both ends of a given peptide can bemodified. The carboxyl and/or amino groups of the carboxyl- andamino-terminal residues, respectively, can be protected or notprotected. The charge at a terminus can also be modified. For example, agroup or radical such as an acyl group (RCO—, where R is an organicgroup (e.g., an acetyl group (CH₃CO—)) can be present at the N-terminusof a peptide to neutralize an “extra” positive charge that may otherwisebe present (e.g., a charge not resulting from the side chain of theN-terminal amino acid). Similarly, a group such as an amine group (RNH—,where R is an organic group (e.g., an amino group —NH₂)) can be used toneutralize an “extra” negative charge that may otherwise be present atthe C-terminus (e.g., a charge not resulting from the side chain of theC-terminal amino acid residue). Where an amine is used, the C-terminusbears an amide (—CONHR). The neutralization of charges on a terminus mayfacilitate self-assembly. One of ordinary skill in the art will be ableto select other suitable groups.

Useful peptides can also be branched, in which case they will contain atleast two peptide “branches”, each of which includes at least threeamino acid residues joined by peptide bonds. The two peptide branchesmay be linked by a bond other than a peptide bond.

The peptides can have an amphiphilic nature (e.g., the peptides cancontain approximately equal numbers of hydrophobic and hydrophilic aminoacid residues), which can be complementary and structurally compatible.Complementary peptides have the ability to form ionic or hydrogen bondswith residues on adjacent peptides in a structure. For example, one ormore hydrophilic residues in a peptide can either hydrogen bond orionically pair with one or more hydrophilic residues on an adjacentpeptide. Hydrophilic residues typically contain a polar functional groupor a functional group that is charged at physiological conditions.Exemplary functional groups include, but are not limited to, carboxylicacid groups, amino groups, sulfate groups, hydroxyl groups, halogengroups, nitro groups, phosphate groups, etc. Hydrophobic residues arethose residues that contain non-polar functional groups. Exemplaryfunctional groups include, but are not limited to, alkyl groups, alkenegroups, alkyne groups, and phenyl groups.

In one embodiment, the hydrophilic residue has the formula—NH—CH(X)—COO—, wherein X has the formula (CH₂)_(y)Z, wherein y=0-8,preferably 1-6, more preferably 1-4 and most preferably 1-3, and Z is apolar or charged functional group including, but not limited to, acarboxylic acid group, an amino group, a sulfate group, a hydroxylgroup, a halogen group, a nitro group, a phosphate group, or afunctional group containing a quaternary amine. The alkyl chain can bein a linear, branched, or cyclic arrangement. X may also contain one ormore heteroatoms within the alkyl chain and/or X may be substituted withone or more additional substituents. In a preferred embodiment, Z is acarboxylic acid group or an amino group. In one embodiment, thehydrophobic residue has the formula —NH—CH(X)—COO—, wherein X has theformula (CH₂)_(y)Z, wherein y=0-8, preferably 1-6, more preferably 1-4,and more preferably 1-3, and Z is a non-polar functional groupincluding, but not limited to, an alkyl group, an alkene group, analkyne group, or a phenyl group. The alkyl, alkene, or alkyne chain canbe in a linear, branched, or cyclic arrangement. X may also contain oneor more heteroatoms within the alkyl chain and/or X may be substitutedwith one or more additional substituents. In a preferred embodiment, Xis an alkyl group, such as a methyl group.

In one embodiment, the SAP includes peptides having a sequence of aminoacid residues conforming to one or more of Formulas I-IV:

((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n);  (I)

((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n);  (II)

((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n);  (III) and

((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n),  (IV)

wherein each Xaa^(neu) represents an amino acid residue having a neutralcharge; Xaa⁺ represents an amino acid residue having a positive charge;Xaa⁻ represents an amino acid residue having a negative charge; x and yare integers having a value of 1, 2, 3, or 4, independently; and n is aninteger having a value of 1-5.

Useful peptides can also include one or more amino acid residue having aneutral charge between one or more sets of residues confirming to anyone of Formulas I-IV. For example, in some embodiments, peptides includeFormulas III and IV, linked with a single amino acid residue having aneutral charge, or linked with two amino acid residues having a neutralcharge, or linked with three amino acid residues having a neutralcharge.

Peptides with modulus I (i.e., peptides having alternate positively andnegatively charged R groups on one side (e.g., the polar face of theβ-sheet) are described by each of Formulas I-IV, where x and y are 1.Examples of peptides of modulus I include, but are not limited to, RADA(SEQ ID NO. 57) and RADARADARADARADA (SEQ ID NO. 1). Examples ofpeptides of modulus II (i.e., peptides having two residues bearing onetype of charge (e.g., a positive charge) followed by two residuesbearing another type of charge (e.g., a neutral charge)) are describedby the same formulas where both x and y are 2. Examples of peptides ofmodulus III (i.e., peptides having three residues bearing one type ofcharge (e.g., a positive charge) followed by three residues bearinganother type of charge (e.g., a negative charge)) include, but are notlimited to, RARARADADADA (SEQ ID NO. 415). Examples of peptides ofmodulus IV (i.e., peptides having four residues bearing one type ofcharge (e.g., a positive charge) followed by four residues bearinganother type of charge (e.g., a negative charge)) include, but are notlimited to, RARARARADADADADA (SEQ ID NO. 416).

In some embodiments, the SAP comprises peptides having a sequence ofamino acid residues comprising one or more of Formulas V-XII:

Xaa^(neu)((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n));  (V)

Xaa^(neu)((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n);  (VI)

((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n)Xaa^(neu);  (VII)

((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n)Xaa^(neu);  (VIII)

((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n)Xaa^(neu);  (IX)

((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n)Xaa^(neu);  (X)

Xaa^(neu)((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n);  (XI)

Xaa^(neu)((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n);  (XII)

Wherein each Xaa^(neu) represents an amino acid residue having a neutralcharge; Xaa⁺ represents an amino acid residue having a positive charge;Xaa⁻ represents an amino acid residue having a negative charge; x and yand z are integers having a value of 1, 2, 3, or 4, independently; and nis an integer having a value of 1-5.

Where SAP are used, it is thought that their side chains (or R groups)partition into two faces, a polar face with positively and/or negativelycharged ionic side chains (e.g., side chains containing —OH, —NH, —CO₂H,or —SH groups), and a nonpolar face with side chains that are consideredneutral or uncharged at physiological pH (e.g., the side chain of analanine residue or residues having other hydrophobic groups). Thepositively charged and negatively charged amino acid residues on thepolar face of one peptide can form complementary ionic pairs withoppositely charged residues of another peptide. These peptides maytherefore be called ionic, self-complementary peptides. If the ionicresidues alternate with one positively and one negatively chargedresidue on the polar face (− + − + − + − +), the peptides may bedescribed as “modulus I;” if the ionic residues alternate with twopositively and two negatively charged residues (− − + + − − + +) on thepolar face, the peptides are described as “modulus II;” if the ionicresidues alternate with three positively and three negatively chargedresidues (+ + + − − − + + + − − −) on the polar face, the peptides aredescribe as “modulus III;” if the ionic residues alternate with fourpositively and four negatively charged residues (+ + + + − − − − + + + +− − − −) on the polar face, they are described as “modulus IV.” Apeptide having four repeating units of the sequence EAKA (SEQ ID NO: 77)may be designated EAKA16-I (SEQ ID NO: 76), and peptides having othersequences may be described by the same convention.

Other hydrophilic residues that form hydrogen bonds including, but notlimited to, asparagine and glutamine, may be incorporated into thepeptides. If the alanine residues in the peptides are changed to morehydrophobic residues, such as leucine, isoleucine, phenylalanine ortyrosine, the resulting peptides have a greater tendency toself-assemble and form peptide matrices with enhanced strength. Somepeptides that have similar amino acid sequences and lengths as thepeptides described form alpha-helices and random-coils, rather thanbeta-sheets, without forming macroscopic structures. In addition toself-complementarity, other factors likely to be important for theformation of macroscopic structures include the peptide length, thedegree of intermolecular interaction, and the ability to form staggeredarrays.

Unpaired residues can interact (e.g., form hydrogen bonds, etc.) withthe solvent. Peptide-peptide interactions may also involve van der Waalsforces and/or forces that do not constitute covalent bonds. The peptidesare structurally compatible when they are capable of maintaining asufficiently constant intrapeptide distance to allow self-assembly andstructure formation. The intrapeptide distance can vary. The term“intrapeptide distance” refers to the average of a representative numberof distances between adjacent amino acid residues. In one embodiment,the intrapeptide distance is less than about 4 angstroms, preferablyless than about 3, more preferably less than about 2 angstroms, and mostpreferably less than about 1 angstrom. The intrapeptide distance may belarger than this, however. These distances can be calculated based onmolecular modeling or based on a simplified procedure described in U.S.Pat. No. 5,670,483 to Zhang, et al.

The compositions including SAP can be formed through self-assembly ofthe peptides described in U.S. Pat. Nos. 5,670,483; 5,955,343;6,548,630; 6,800,481; 7,098,028; 9,327,010; and U.S. Pat. No. 9,364,513to Zhang, et al.; U.S. Pat. Nos. 9,162,005; 9,415,084; and U.S. Pat. No.9,339,476 to Ellis-Behnke, et al.; Holmes, et al., Proc. Natl. Acad.Sci. USA, 97:6728-6733 (2000); Zhang, et al., Proc. Natl. Acad. Sci.USA, 90:3334-3338 (1993); Zhang, et al., Biomaterials, 16:1385-1393(1995); Caplan et al., Biomaterials, 23:219-227 (2002); Leon, et al., J.Biomater. Sci. Polym. Ed., 9:297-312 (1998); and Caplan, et al.,Biomacromolecules, 1:627-631 (2000). See also WO 2007/142757.

In some embodiments, SAP include one or more segments of consecutivepositively or negatively charged residues. For example, these segmentscan include a sequence of positively or negatively charged residues, forexample, about 2 to about 50 amino acid residues, typically about 3 toabout 30 residues, more typically about 10 to about 20 amino acidresidues. In some embodiments, about half of the residues of an SAP canbe positively charged and about half of the residues can be negativelycharged. For example, an SAP can have the following sequence RRRRDDDD(SEQ ID NO: 417) or GGGGSSSS (SEQ ID NO: 418). A combination of thesepeptides can self-assemble by matching or aligning the positive end of afirst SAP to the negative end of a second self-assembling peptide. TheSAP can stack-up or aggregate.

In some embodiments, an SAP can contain a segment of residues that haveeither a positive or negative charge under physiological conditions. Forexample, representative amino acid sequences for positively charged SAPinclude, but are not limited to, KKKK (SEQ ID NO: 419), RRRR (SEQ ID NO:420), or HHHH (SEQ ID NO: 421). Representative amino acid sequences fornegatively charged SAP include, but are not limited to, DDDD (SEQ ID NO:422) or EEEE (SEQ ID NO: 423). When combined, a string of positivelycharged amino acid residues align parallel and opposite with a string ofnegatively charged amino acid residues. In certain embodiments, stringsof positively and negatively charged amino acids will alternate and forma multilayered structure.

In some embodiments, an SAP can contain sequences in which at least onehydrophobic residue alternates with at least one hydrophilic residue(under physiological conditions). For example, the sequence of arepresentative SAP can be GQGQ (SEQ ID NO: 424), GGQQGG (SEQ ID NO:425), GQQGQQG (SEQ ID NO: 426), GGQGGQGG (SEQ ID NO: 427), etc.

The partitioning of the SAP into a non-polar or polar environment can becontrolled by altering hydrophobic to hydrophilic amino acid residueratio, wherein a ratio greater than 1:1 indicates that the peptidepartitions more in hydrophobic conditions, while a ratio of less than1:1 indicates that the peptide partitions more in hydrophilic conditions

The compositions, regardless of the precise form (e.g., whether in aliquid form or molded) and regardless of the overall compositions (e.g.,whether combined with another agent, contained within a device, orpackaged in a kit), can include a mixture of one or more peptides.Peptide-based structures can be formed of heterogeneous mixtures ofpeptides (i.e., mixtures containing more than one type of peptideconforming to a given formula or to two or more of the formulas). Insome embodiments, each of the types of peptides in the mixture canself-assemble with the same type of peptide. In other embodiments, oneor more of each type of peptide would not self-assemble alone, but thecombination of heterogeneous peptides may self-assemble (i.e., peptidesin the mixture are complementary and structurally compatible with eachother). Thus, either a homogeneous mixture of self-complementary andself-compatible peptides of the same sequence or containing the samerepeating subunit, or a heterogeneous mixture of different peptides,which are complementary and structurally compatible to each other, canbe used.

In some embodiments, mixtures of one or more peptide sequences producestructures having combined properties of the different sequences used.The physical properties of self-assembled peptide structures varyaccording to the ratio of the different SAP from which they are formed.

In some embodiments, SAP structures include two or more layers ofstructurally distinct SAP structures, for example, formed by consecutiveadministration and assembly of each peptide onto the surface of theother. Therefore, in some embodiments, the structural and biochemicalproperties of each surface of a multi-layered SAP structure aredifferent, according to the different properties of the SAP from whichthey are formed, respectively.

One or more short amino acid sequences that assist in self-assembly(referred to as assembly assist sequences) can be added to a homogeneousor heterogeneous mixture of amino acid sequences that alone do notself-assemble. The assembly assist sequences contain amino acids thatare complementary with the amino acids in the sequences in the mixture.The assembly assist sequences may contain any number of amino acids.Preferably, the assembly assist sequences contain at least four aminoacids. The assembly assist sequences may contain a flexible linkerbetween the amino acids that assists in self-assembly. For example, theassembly assist sequence may contain a pair, a triad, or a quartet ofassembly assisting amino acids at the termini connected via a flexiblelinker. Suitable assembly assist sequences include, but are not limitedto, RADA (SEQ ID NO: 57) and EAKA (SEQ ID NO: 77).

Suitable linkers include, but are not limited to, ether based tetherssuch as polyethylene glycol (PEG), N-succinimidyl3-(2-pyridyldithio)propionate (SPDP, 3- and 7-atom spacer),long-chain-SPDP (12-atom spacer),(succinimidyloxycarbonyl-α-methyl-2-(2-pyridyldithio) toluene) (SMPT,8-atom spacer),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (SMCC,11-atom spacer) andsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,(sulfo-SMCC, 11-atom spacer), m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS, 9-atom spacer), N-(γ-maleimidobutyryloxy) succinimide ester(GMBS, 8-atom spacer), N-(γ-maleimidobutyryloxy) sulfosuccinimide ester(sulfo-GMBS, 8-atom spacer), succinimidyl 6-((iodoacetyl) amino)hexanoate (SIAX, 9-atom spacer), succinimidyl6-(6-(((4-iodoacetyl)amino)hexanoyl)amino)hexanoate (SIAXX, 16-atomspacer), and p-nitrophenyl iodoacetate (NPIA, 2-atom spacer). One ofordinary skill in the art also will recognize that a number of otherlinkers with varying numbers of atoms may be used.

SAP structures can be formed that have varying degrees of stiffness orelasticity. The structures typically have a low elastic modulus (e.g., amodulus in the range of between about 0.01 and about 1,000 kPa,preferably between about 1 and about 100 kPa, more preferably betweenabout 1 and about 10 kPa as measured by standard methods, such as in astandard cone-plate rheometer). Low values may be preferable, as theypermit structure deformation as a result of movement, in response topressure, for instance in the event of cell contraction. Stiffness canbe controlled in a variety of ways, including by changing the length,sequence, and/or concentration of the precursor molecules (e.g., SAP).Other methods for increasing stiffness can also be employed. Forexample, one can attach to the precursors either biotin or othermolecules that can be subsequently cross-linked or otherwise bonded toone another. The molecules (e.g., biotin) can be included at an N- orC-terminus of a peptide/peptidomimetic or attached to one or moreresidues between the termini. Where biotin is used, cross-linking can beachieved by subsequent addition of avidin. Other cross-linkablemolecules can be used, for example, amino acid residues withpolymerizable groups such as vinyl groups may be incorporated andcross-linked by exposure to UV light. The extent of crosslinking can beprecisely controlled by applying the radiation for a predeterminedlength of time. The extent of crosslinking can be determined by lightscattering, gel filtration, scanning electron microscopy, or othermethods well known in the art. Crosslinking can be assessed by HPLC ormass spectrometry analysis of the structure after digestion with aprotease, such as a matrix metalloprotease. Material strength may bedetermined before and/or after cross-linking. Regardless of whethercross-linking is achieved by a chemical agent or light energy, themolecules may be cross-linked in the course of creating a mold or whenpeptide-containing solutions are applied to the eye.

SAP chains can be cross-linked (e.g., to form a spider web-type pattern)to reinforce the material in vivo. The crosslinks can serve to reinforcethe material to provide increased rigidity and strength. For example, anSAP functionalized with a polymerizable group at the periphery can beapplied to the surface of the eye. Upon crosslinking, the peripheralmaterial becomes more rigid, anchoring the material to the surface ofthe eye or other ocular tissue while the interior material remainsflexible to move with the body tissue.

Factors influencing the physical properties of self-assembled peptidestructures at the surface of the eye include, but are not limited to,peptide sequence, peptide length, presence of bound agents, presence oftissue-specific or tissue-binding motifs, for example, a tissue-specificpeptide sequence, as well as peptide amount (e.g., concentration, massand volume), peptide form (e.g., powder or solution) and assembly-stateat application time.

The half-life (e.g., the in vivo half-life) of the structures formed bySAP can also be modulated by incorporating protease or peptidasecleavage sites into the precursors that subsequently form a givenstructure. Proteases or peptidases that occur naturally in vivo or thatare administered can promote degradation by cleaving their cognatesubstrates.

Combinations of any of the modifications here can be made. For example,SAP that include a protease cleavage site and a cysteine residue and/ora cross-linking agent, kits and devices containing them, and methods ofusing them, can be utilized.

The peptide structures formed from any SAP made by any process can becharacterized using various biophysical and optical techniques, such ascircular dichroism (CD), dynamic light scattering, Fourier transforminfrared (FTIR), atomic foRCES (tension) microscopy (ATM), scanningelectron microscopy (SEM), and transmission electron microscopy (TEM).For example, biophysical methods can be used to determine the degree ofbeta-sheet secondary structure in the peptide structure. Filament andpore size, fiber diameter, length, elasticity, and volume fraction canbe determined using quantitative image analysis of scanning and/ortransmission electron micrographs. The structures can also be examinedusing several standard mechanical testing techniques to measure theextent of swelling, the effect of pH and ion concentration on structureformation, the level of hydration under various conditions, the tensilestrength, as well as the manner in which various characteristics changeover the period of time required for the structures to form and degrade.Typically, the SAP are biocompatible, non-toxic, fully or partiallybiodegradable, and do not cause local or systemic inflammation.Preferably, break down products of the SAP do not cause secondarytoxicity and are preferably suitable for growth and repair of thesurrounding tissues.

2. Peptidomimetics

Another class of materials that can self-assemble is peptidomimetics.The term “peptidomimetics” refers to non-natural peptide molecules whichmimic peptide structure. Peptidomimetics typically retain the ability toproduce the same biological effect as a parent peptide and can interactwith the biological target of the parent peptide. Peptidomimetics may beused to circumvent some of the problems associated with a naturalpeptide: e.g. stability against proteolysis and poor bioavailability(Vagner J., et al. Curr. Opin. Chem. Biol., 12(3): 292-296 (2008)).Self-assembling eptidomimetics are molecules that are structurallysimilar to peptides, having a segment of residues having a positivecharge under physiological conditions joined to a segment of residueshaving a negative charge under physiological conditions.

Peptidomimetics have general features analogous to peptides, such asamphiphilicity. Examples of peptidomimetics are described in Moore etal., Chem. Rev. 101(12), 3893-4012 (2001), and in WO 2007/142757

The peptidomimetics can be classified into four categories: α-peptides,β-peptides, γ-peptides, and δ-peptides. Peptides including combinationsof more than one of α-amino acids, β-amino acids, γ-amino acids, andδ-amino acids can also be used. For example, SAP includes alpha-aminoand beta-amino acid residues (i.e., alpha-beta peptides), alpha-aminoand delta-amino acid residues (i.e., alpha-delta peptides), andalpha-amino and gamma-amino acid residues (i.e., alpha-gamma peptides).

The alpha amino acids can be classical or non-classical alpha aminoacids (i.e., L-form or D-form, or combinations thereof). Examples ofα-peptide peptidomimetics that can be used include N,N′-linkedoligoureas, oligopyrrolinones, oxazolidin-2-ones, azatides andazapeptides.

Examples of β-peptides include β-peptide foldamers, β-aminoxy acids,sulfur-containing β-peptide analogues, and hydrazino peptides.

Examples of γ-peptides include γ-peptide foldamers, oligoureas,oligocarbamates, and phosphodiesters.

Examples of δ-peptides include alkene-based δ-amino acids andcarbopeptoids, such as pyranose-based carbopeptoids and furanose-basedcarbopeptoids.

SAP can be generated, for example, which differ from those exemplifiedby a single amino acid residue or by multiple amino acid residues (e.g.,by inclusion or exclusion of a repeating quartet). For example, one ormore cysteine residues may be incorporated into the peptides, and theseresidues may bond with one another through the formation of disulfidebonds. Structures bonded in this manner may have increased mechanicalstrength relative to structures made with comparable peptides that donot include cysteine residues and thus are unable to form disulfidebonds.

3. Exemplary SAP

In an exemplary embodiment, a self-assembling peptidomimetic includesboth alpha amino acids (annotated as Xaa) and beta amino acids(annotated as Xaa^(B)). Exemplary self-assembling peptidomimeticsequences include EA^(B)KA^(B)EA^(B)KA^(B)EA^(B)KA^(B)EA^(B)KA^(B) (SEQID NO: 428); EA^(B)KA^(B)EA^(B)KA^(B) (SEQ ID NO: 429);RA^(B)DA^(B)RA^(B)DA^(B)RA^(B)DA^(B)RA^(B)DA^(B) (SEQ ID NO: 430); andRA^(B)DA^(B)RA^(B)DA^(B) (SEQ ID NO: 431).

Examples of representative hydrophobic and hydrophilic SAP sequences arelisted in Table 1.

TABLE 1 Representative SAP Sequence (N→C) SEQ ID NO: RADARADARADARADA  1SGSGSGSGSGSGSGSG  2 SASASASASASASASA  3 SVSVSVSVSVSVSVSV  4SLSLSLSLSLSLSLSL  5 SISISISISISISISI  6 SMSMSMSMSMSMSMSM  7SFSFSFSFSFSFSFSF  8 SWSWSWSWSWSWSWSW  9 SPSPSPSPSPSPSPSP 10TGTGTGTGTGTGTGTG 11 TATATATATATATATA 12 TVTVTVTVTVTVTVTV 13TLTLTLTLTLTLTLTL 14 TITITITITITITITI 15 TMTMTMTMTMTMTMTM 16TFTFTFTFTFTFTFTF 17 TWTWTWTWTWTWTWTW 18 TPTPTPTPTPTPTPTP 19CGCGCGCGCGCGCGCG 20 CACACACACACACACA 21 CVCVCVCVCVCVCVCV 22CLCLCLCLCLCLCLCL 23 CICICICICICICICI 24 CMCMCMCMCMCMCMCM 25CFCFCFCFCFCFCFCF 26 CWCWCWCWCWCWCWC 27 CPCPCPCPCPCPCPCP 28YGYGYGYGYGYGYGYG 29 YAYAYAYAYAYAYAYA 30 YVYVYVYVYVYVYVYV 31YLYLYLYLYLYLYLYL 32 YIYIYIYIYIYIYIYI 33 YMYMYMYMYMYMYMYM 34YFYFYFYFYFYFYFYF 35 YWYWYWYWYWYWYWYW 36 YPYPYPYPYPYPYPYP 37NGNGNGNGNGNGNGNG 38 NANANANANANANANA 39 NVNVNVNVNVNVNVNV 40NLNLNLNLNLNLNLNL 41 NINININININININI 42 NMNMNMNMNMNMNMNM 43NFNFNFNFNFNFNFNF 44 NWNWNWNWNWNWNWNW 45 NPNPNPNPNPNPNPNP 46QGQGQGQGQGQGQGQG 47 QAQAQAQAQAQAQAQA 48 QVQVQVQVQVQVQVQV 49QLQLQLQLQLQLQLQL 50 QIQIQIQIQIQIQIQI 51 QMQMQMQMQMQMQMQM 52QFQFQFQFQFQFQFQF 53 QWQWQWQWQWQWQWQW 54 QPQPQPQPQPQPQPQP 55AEAKAEAKAEAKAEAK 56 RADA 57 RAEARAEARAEARAEA 58 KADAKADAKADAKADA 59ARADARADARADA 60 RADARADARADARADARADA 61 ARADARADARADARADARADA 62ARADARADARADARADA 63 RLDLRLDLRLDLRLDL 64 RLDL 65 RLDLRL 66 RADARA 67LRLDLR 68 IEIKIEIKIEIKI 69 IEIKIEIKIEIKIEIK 70 IEIKIEIKIEIKIEIKI 71IEIKIEIKIEIKIEIKIEIK 72 IEIKIEIKIEIKIEIKIEIKI 73 IEIKIEIKIEIK 74EIKIEIKIEIKIEIKI 75 EAKAEAKAEAKAEAKA 76 EAKA 77 EAKAEAKAEA 78EAKAEAKAEAKAEAKAEAKA 79 AEAKAEAKAEAKAEAKA 80 AEAKAEAKAEAKA 81RADARADARADARADLRA-c 82R^(D)A^(D)D^(D)A^(D)R^(D)A^(D)D^(D)A^(D)R^(D)A^(D)D^(D)A^(D)R^(D)A^(D)D^(D)A^(D)83 R^(D)A^(D)D^(D)A^(D)R^(D)A^(D)D^(D)A^(D)R^(D)A^(D)D^(D)A^(D) 84E^(D)A^(D)K^(D)A^(D)E^(D)A^(D)K^(D)A^(D)E^(D)A^(D)K^(D)A^(D) 85E^(D)A^(D)K^(D)A^(D) 86 R^(D)A^(D)D^(D)A^(D) 87 RADARADA 88EARAEARAEARAEARA 89 EARAEARAEARA 90 EARAEARAE 91 EARA 92

B. Tissue-Specific Components

The SAP may contain a tissue-specific component (“TSC”), which can bepeptides, polysaccharides, or glycoproteins that are present within theeye, or that are specific to the tissue surrounding or in contact withthe eye. Specificity can vary in degree. For example, a TSC may bind toa single cell type or to cells found in one type of tissue, inconnective tissue, to epithelial cells, or by species (human) cells, orspecific organs or organelles.

TSC bind to tissues within or adjacent to the ocular compartmentincluding tissues at or near the surface of the eye, tissues within theinterior cavities of the eye, and tissue surrounding the eye. Tissues ator near the surface of the eye include the tissues of the cornealepithelium, the Bowman's membrane layer, the corneal stroma, theDescemet's membrane of the cornea, the corneal endothelium, the fornixconjunctiva, the scleral conjunctiva, the bulbar conjunctiva, themarginal conjunctiva, the tarsal conjunctiva, the orbital conjunctivaand the limbal conjunctiva. Exemplary tissues within the interior of theeye include tissues of the anterior cavity, the scleral venous sinus,the lens, the suspensory ligament of the lens, the vitreous chamber, theretina, the ciliary cavity, the retina, the retinal arteries and veins,the optic disc and the choroid. Exemplary tissues surrounding the eyeinclude the palpebral conjunctiva, the tear ducts, tarsal glands, thelacus lacrimalis, the lacrimal punctum, and the epithelial tissueslining the interior of the ocular cavity.

The TSC can target cell specific surface carbohydrates. Celltype-specific carbohydrates are involved in cell-cell interactions. Insome embodiments, the TSC is a sequence of amino acids that recognizesand interacts with one or more components of injured or diseased tissue.In some embodiments, TSC interact with a ligand or component that iscommon to many tissues, and that is also expressed in the eye. In otherembodiments, the TSC interacts with a sequence that is not present orexposed in healthy tissue. In certain embodiments, the TSC interactswith one or more of the components of the extracellular matrix (ECM).The SAP can be modified such that they can anchor or interact with thestructural ECM at the edges of blood vessels and/or tissues. ECM is anymaterial part of a tissue that is not part of any cell, and it is thedefining feature of connective tissue. The ECM's main components arevarious glycoproteins, proteoglycans and hyaluronic acid. In mostanimals, the most abundant glycoproteins in the ECM are collagens. ECMalso contains many other components: proteins such as fibrin, elastin,fibronectins, laminins, and nidogens, and minerals such ashydroxyapatite, or fluids such as blood plasma or serum with secretedfree flowing antigens. In addition, the ECM sequesters a wide range ofcellular growth factors, and acts as a local depot for them. Changes inphysiological conditions can trigger protease activities that causelocal release of such depots. This allows the rapid and local activationof cellular functions, without de novo synthesis. Given this diversity,ECM can serve many functions, such as providing support and anchoragefor cells, providing a way of separating the tissues, and regulatingintercellular communication.

1. TSC Sequences

Peptides or proteins can be used in combination with or alternating withthe SAP. Representative TSC are provided in Table 2.

TABLE 2 Tissue Specific Components Sequence (N→C) SEQ ID NO:Pmp(Y(Me)ITNCP-Orn-Y)NH₂ 409 Mpr(YFQNCPR) 410 (CYFQNCPRG)NH₂ 411CYFQNCPR 412 (CYIQNCPRG)NH₂  93 (YFQN(Asu)PRG)NH₂  94 (YIQN(Asu)PRG)NH₂ 95 Mpr-D-  96 PyridylAnine(FQNCPRG)NH₂ (Deamino-Pen-YFVNCPDRG)NH₂  97Mpr(YFQNCPRG)NH₂  98 Mpr(YFQNCPDRG)NH₂  99 Mpr(YFQNCPK) 100(CYFQNCPKG)NH₂ 101 CYFQNCPK 102 Mpr(YFVNCPDRG)NH₂ 103 (CFIQNCP-Orn-G)NH₂104 Pmp(DY(OEt)FVNCP-Cit-G)NH₂ 105 Pmp(Y(OEt)FVNCPRG)NH₂ 106Pmp(Y(Me)FQNCPRG)NH₂ 107 Pmp(Y(Me)IQNCP-Orn-G)NH₂ 108 GDRGDSP 109GDRGDSPASSK 110 G-Pen-GRGDSPCA 111 GRADSP 112 GRGDDSP 113 GRGDNP 114GRGDS 115 GRGDSP 116 GRGDSPC 117 GRGDSPK 118 GRGDTP 119 GRGES 120 GRGESP121 GRGETP 122 KGDS 123 GAVSTA 124 WTVPTA 125 TDVNGDGRHDL 126 REDV 127RGDC 128 RGDS 129 RGDSPASSKP 130 RGDT 131 RGDV 132 RGES 133 SDGR 134SDGRG 135 YRGDS 136 EGVNDNEEGFFSAR 137 YADSGEGDFLAEGGGVR 138G(Glp)VNDNEEGFFSARY 139 GPR N/A MSCRAMM 141 Pmp = pyridoxamine phosphateMpr = 3-mercaptopropionyl Deamino-Pen = deamino penicillamine Pen =penicillamine Asu = amino succinyl OEt = ethoxy Me = methyl Cit =citrulline

C. Hydrophobic Peptide Sequences

Hydrophobic or hydrophilic tails can be added to the SAP. The tails caninteract with cell membranes, thus anchoring the SAP on to the cellsurface. Table 3 shows a list of peptides with hydrophobic tails.

TABLE 3 SAP including Hydrophobic Tails Sequence (N→ C) SEQ ID NO:GGGGGDGDGDGDGDGD 142 GGGGGEGEGEGEGEGE 143 GGGGGKGKGKGKGKGK 144GGGGGRGRGRGRGRGR 145 GGGGGHGHGHGHGHGH 146 AAAAADADADADADAD 147AAAAAEAEAEAEAEAE 148 AAAAAKAKAKAKAKAK 149 AAAAARARARARARAR 150AAAAAHAHAHAHAHAH 151 VVVVVDVDVDVDVDVD 152 VVVVVEVEVEVEVEVE 153VVVVVKVKVKVKVKVK 154 VVVVVRVRVRVRVRVR 155 VVVVVHVHVHVHVHVH 156LLLLLDLDLDLDLDLD 157 LLLLLELELELELELE 158 LLLLLKLKLKLKLKLK 159LLLLLRLRLRLRLRLR 160 LLLLLHLHLHLHLHLH 161 IIIIIDIDIDIDIDID 162IIIIIEIEIEIEIEIE 163 IIIIIKIKIKIKIKIK 164 IIIIIRIRIRIRIRIR 165IIIIIHIHIHIHIHIH 166 MMMMMDMDMDMDMDMD 167 MMMMMEMEMEMEMEME 168MMMMMKMKMKMKMKMK 169 MMMMMRMRMRMRMRMR 170 MMMMMHMHMHMHMHMH 171FFFFFDFDFDFDFDFD 172 FFFFFEFEFEFEFEFE 173 FFFFFKFKFKFKFKFK 174FFFFFRFRFRFRFRFR 175 FFFFFHFHFHFHFHFH 176 WWWWWDWDWDWDWDWD 177WWWWWEWEWEWEWEWE 178 WWWWWKWKWKWKWKWK 179 WWWWWRWRWRWRWRWR 180WWWWWHWHWHWHWHWH 181 PPPPPDPDPDPDPDPD 182 PPPPPEPEPEPEPEPE 183PPPPPKPKPKPKPKPK 184 PPPPPRPRPRPRPRPR 185 PPPPPHPHPHPHPHPH 186AAAAARADARADARAD 187 AAAAARARADADARAR 188 AAAAAEAKAEAKAEAK 189AAAAAEAEAKAKAEAE 190 AAAAARAEARAEARAE 191 AAAAARARAEAEARAE 192AAAAAKADAKADAKAD 193 AAAAAEAHAEAHAEAH 194 AAAAAEAEAHAHAEAE 195AAAAARARARARADAD 196 AAAAARARARADADAD 197 AAAAAHADAHADAHAD 198AAAAAHADADAHADAD 199 AAAAAHAEAEAHAEAE 200 GGGGGRGDGRGDGRGD 201GGGGGRGRGDGDGRGR 202 GGGGGEGKGEGKGEGK 203 GGGGGEGEGKGKGEGE 204GGGGGRGEGRGEGRGE 205 GGGGGRGRGEGEGRGE 206 GGGGGKGDGKGDGKGD 207GGGGGEGHGEGHGEGH 208 GGGGGEGEGHGHGEGE 209 GGGGGRGRGRGRGDGD 210GGGGGRGRGRGDGDGD 211 GGGGGHGDGHGDGHGD 212 GGGGGHGDGDGHGDGD 213GGGGGHGEGEGHGEGE 214 VVVVVRVDVRVDVRVD 215 VVVVVRVRVDVDVRVR 216VVVVVEVKVEVKVEVK 217 VVVVVEVEVKVKVEVE 218 VVVVVRVEVRVEVRVE 219VVVVVRVRVEVEVRVE 220 VVVVVKVDVKVDVKVD 221 VVVVVEVHVEVHVEVH 222VVVVVEVEVHVHVEVE 223 VVVVVRVRVRVRVDVD 224 VVVVVRVRVRVDVDVD 225VVVVVHVDVHVDVHVD 226 VVVVVHVDVDVHVDVD 227 VVVVVHVEVEVHVEVE 228LLLLLRLDLRLDLRLD 229 LLLLLRLRLDLDLRLR 230 LLLLLELKLELKLELK 231LLLLLELELKLKLELE 232 LLLLLRLELRLELRLE 233 LLLLLRLRLELELRLE 234LLLLLKLDLKLDLKLD 235 LLLLLELHLELHLELH 236 LLLLLELELHLHLELE 237LLLLLRLRLRLRLDLD 238 LLLLLRLRLRLDLDLD 239 LLLLLHLDLHLDLHLD 240LLLLLHLDLDLHLDLD 241 LLLLLHLELELHLELE 242 IIIIIRIDIRIDIRID 243IIIIIRIRIDIDIRIR 244 IIIIIEIKIEIKIEIK 245 IIIIIEIEIKIKIEIE 246IIIIIRIEIRIEIRIE 247 IIIIIRIRIEIEIRIE 248 IIIIIKIDIKIDIKID 249IIIIIEIHIEIHIEIH 250 IIIIIEIEIHIHIEIE 251 IIIIIRIRIRIRIDID 252IIIIIRIRIRIDIDID 253 IIIIIHIDIHIDIHID 254 IIIIIHIDIDIHIDID 255IIIIIHIEIEIHIEIE 256 MMMMMRMDMRMDMRMD 257 MMMMMRMRMDMDMRMR 258MMMMMEMKMEMKMEMK 259 MMMMMEMEMKMKMEME 260 MMMMMRMEMRMEMRME 261MMMMMRMRMEMEMRME 262 MMMMMKMDMKMDMKMD 263 MMMMMEMHMEMHMEMH 264MMMMMEMEMHMHMEME 265 MMMMMRMRMRMRMDMD 266 MMMMMRMRMRMDMDMD 267MMMMMHMDMHMDMHMD 268 MMMMMHMDMDMHMDMD 269 MMMMMHMEMEMHMEME 270FFFFFRFDFRFDFRFD 271 FFFFFRFRFDFDFRFR 272 FFFFFEFKFEFKFEFK 273FFFFFEFEFKFKFEFE 274 FFFFFRFEFRFEFRFE 275 FFFFFRFRFEFEFRFE 276FFFFFKFDFKFDFKFD 277 FFFFFEFHFEFHFEFH 278 FFFFFEFEFHFHFEFE 279FFFFFRFRFRFRFDFD 280 FFFFFRFRFRFDFDFD 281 FFFFFHFDFHFDFHFD 282FFFFFHFDFDFHFDFD 283 FFFFFHFEFEFHFEFE 284 WWWWWRWDWRWDWRWD 285WWWWWRWRWDWDWRWR 286 WWWWWEWKWEWKWEWK 287 WWWWWEWEWKWKWEWE 288WWWWWRWEWRWEWRWE 289 WWWWWRWRWEWEWRWE 290 WWWWWKWDWKWDWKWD 291WWWWWEWHWEWHWEWH 292 WWWWWEWEWHWHWEWE 293 WWWWWRWRWRWRWDWD 294WWWWWRWRWRWDWDWD 295 WWWWWHWDWHWDWHWD 296 WWWWWHWDWDWHWDWD 297WWWWWHWEWEWHWEWE 298 PPPPPRPDPRPDPRPD 299 PPPPPRPRPDPDPRPR 300PPPPPEPKPEPKPEPK 301 PPPPPEPEPKPKPEPE 302 PPPPPRPEPRPEPRPE 303PPPPPRPRPEPEPRPE 304 PPPPPKPDPKPDPKPD 305 PPPPPEPHPEPHPEPH 306PPPPPEPEPHPHPEPE 307 PPPPPRPRPRPRPDPD 308 PPPPPRPRPRPDPDPD 309PPPPPHPDPHPDPHPD 310 PPPPPHPDPDPHPDPD 311 PPPPPHPEPEPHPEPE 312SSSSSRSDSRSDSRSD 313 SSSSSRSRSDSDSRSR 314 SSSSSESKSESKSESK 315SSSSSESESKSKSESE 316 SSSSSRSESRSESRSE 317 SSSSSRSRSESESRSE 318SSSSSKSDSKSDSKSD 319 SSSSSESHSESHSESH 320 SSSSSESESHSHSESE 321SSSSSRSRSRSRSRSR 322 SSSSSRSRSRSRSDSD 323 SSSSSRSRSRSDSDSD 324SSSSSHSDSHSDSHSD 325 SSSSSHSHSHSHSHSH 326 SSSSSHSDSDSHSDSD 327SSSSSHSESESHSESE 328 TTTTTRTDTRTDTRTD 329 TTTTTRTRTDTDTRTR 330TTTTTETKTETKTETK 331 TTTTTETETKTKTETE 332 TTTTTRTETRTETRTE 333TTTTTRTRTETETRTE 334 TTTTTKTDTKTDTKTD 335 TTTTTETHTETHTETH 336TTTTTETETHTHTETE 337 TTTTTRTRTRTRTRTR 338 TTTTTRTRTRTRTDTD 339TTTTTRTRTRTDTDTD 340 TTTTTHTDTHTDTHTD 341 TTTTTHTHTHTHTHTH 342TTTTTHTDTDTHTDTD 343 TTTTTHTETETHTETE 344 CCCCCRCDCRCDCRCD 345CCCCCRCRCDCDCRCR 346 CCCCCECKCECKCECK 347 CCCCCECECKCKCECE 348CCCCCRCECRCECRCES 349 CCCCCRCRCECECRCES 350 CCCCCKCDCKCDCKCD 351CCCCCECHCECHCECH 352 CCCCCECECHCHCECE 353 CCCCCRCRCRCRCRCR 354CCCCCRCRCRCRCDCD 355 CCCCCRCRCRCDCDCD 356 CCCCCHCDCHCDCHCD 357CCCCCHCHCHCHCHCH 358 CCCCCHCDCDCHCDCD 359 CCCCCHCECECHCECE 360YYYYYRYDYRYDYRYD 361 YYYYYRYRYDYDYRYR 362 YYYYYEYKYEYKYEYK 363YYYYYEYEYKYKYEYE 364 YYYYYRYEYRYEYRYE 365 YYYYYRYRYEYEYRYE 366YYYYYKYDYKYDYKYD 367 YYYYYEYHYEYHYEYH 368 YYYYYEYEYHYHYEYE 369YYYYYRYRYRYRYRYR 370 YYYYYRYRYRYRYDYD 371 YYYYYRYRYRYDYDYD 372YYYYYHYDYHYDYHYD 373 YYYYYHYHYHYHYHYH 374 YYYYYHYDYDYHYDYD 375YYYYYHYEYEYHYEYE 376 NNNNNRNDNRNDNRND 377 NNNNNRNRNDNDNRNR 378NNNNNENKNENKNENK 379 NNNNNENENKNKNENE 380 NNNNNRNENRNENRNE 381NNNNNRNRNENENRNE 382 NNNNNKNDNKNDNKND 383 NNNNNENHNENHNENH 384NNNNNENENHNHNENE 385 NNNNNRNRNRNRNRNR 386 NNNNNRNRNRNRNDND 387NNNNNRNRNRNDNDND 388 NNNNNHNDNHNDNHND 389 NNNNNHNHNHNHNHNH 390NNNNNHNDNDNHNDND 391 NNNNNHNENENHNENE 392 QQQQQRQDQRQDQRQD 393QQQQQRQRQDQDQRQR 394 QQQQQEQKQEQKQEQK 395 QQQQQEQEQKQKQEQE 396QQQQQRQEQRQEQRQE 397 QQQQQRQRQEQEQRQE 398 QQQQQKQDQKQDQKQD 399QQQQQEQHQEQHQEQH 400 QQQQQEQEQHQHQEQE 401 QQQQQRQRQRQRQRQR 402QQQQQRQRQRQRQDQD 403 QQQQQRQRQRQDQDQD 404 QQQQQHQDQHQDQHQD 405QQQQQHQHQHQHQHQH 406 QQQQQHQDQDQHQDQD 407 QQQQQHQEQEQHQEQE 408

Hydrophilic tails can be added to the SAP, alone or in addition tohydrophobic tails, to facilitate interaction with the ECM of differentvessels or tissues, such as the bladder.

D. Formulations of SAPs

The compositions can be used to prevent or limit movement of a bodilyfluid, to stabilize tissue, components thereof or cells, or to preventcontamination when administered to a site in need thereof. Thecompositions can be in the form of a dry powder, a wafer, a disk, atablet, a capsule, a liquid, a gel, a cream, a foam, an ointment, anemulsion, a coating on a medical device or implant, incorporated into amicroparticle, a polymeric matrix, a hydrogel, a fabric, a bandage, asuture, a contact lens, an eye drop, an eye patch, or a sponge.

The concentration of the SAP in any given formulation can vary and canbe between approximately 0.1% and 99%, inclusive, preferably between0.1% and 10%. In one embodiment, the concentration of the SAP in aliquid formulation is between approximately 0.1 and 10.0% (1-100 mg/ml).The concentration of SAP can be higher in stock solutions and in solid(e.g., powdered) formulations. Solid preparations may have aconcentration of SAP approaching 100% (e.g., the concentration of SAPcan be 75, 80, 85, 90, 95, 96, 97, 98, 99% or more (e.g., 99.99%) of thecomposition).

In some embodiments, compositions of SAP are formulated for applicationto the eye as a dry powder. Dry powder formulation may contain at least75% weight/weight (w/w) SAP, at least 80% w/w, at least 85% w/w, atleast 90% w/w, at least 95% w/w, or more than 95% w/w.

In other embodiments, the SAP are formulated for application to the eyeas a solution. Based on studies using RADA (SEQ ID NO: 57) and EAKA (SEQID NO: 77), SAP can be can be present in an aqueous solution thatcontains from about 0.25% weight/volume (w/v), to at least 7.5% w/v,preferably from about 1% w/v, to about 6% w/v, inclusive, for example,at least 0.1%, such as 0.1%-1%, 0.5%-5%, 1%-4%, 1%-5%, 1%-6%, 2%, 3%, 4%or 5% w/v. In some embodiments, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or more than 95% of the SAP have thesame size and sequence. In particular embodiments the SAP include two ormore repeating units of the sequence RADA (SEQ ID NO: 57), two or morerepeating units of the sequence EAKA (SEQ ID NO: 77), or combinationsthereof.

In some forms, compositions of SAP formulated for application onto thesurface of the eye are preferably of low viscosity, and flow across theentire surface of the eye to form a continuous SAP structure (e.g., one“layer”) across the exposed surface of the cornea. The concentration ofSAP in the formulations for application to the surface of the eyetypically are between about 0.05% and about 0.5% w/v, for example,0.1%-0.3% of the solution, in an amount sufficient to coat the entireexposed surface of the cornea. An exemplary volume for application tothe surface of the eye is between about 10 μl and about 1,000 μl.

The viscosity of the SAP formulation may be adjusted to mimic that of abodily fluid, for example, the vitreous humor. In other embodiments, theSAP forms a barrier to movement of fluids and bodily substances, forexample, to prevent or reduce cell-cell interactions and cell-cellsignaling within or at the surface of the eye. In these embodiments, theconcentration of the SAP within the solution is between about 1% andabout 4% w/v, for example, 2.5% w/v. Further, the density of thematerial may be adjusted or sufficiently raised such that it maintainsenhanced resting contact with a healing retina, thereby allowing thepatient greater freedom of head movement of the head.

Assembly of the SAP can be initiated or enhanced by the addition of anionic solute or diluent to a solution of the SAP or by a change in pH.For example, NaCl at a concentration of at least 5 mM can induce theassembly of macroscopic structures within a short period of time (e.g.,within a few seconds to minutes). Lower concentrations of NaCl may alsoinduce assembly but at a slower rate. Alternatively, self-assembly maybe initiated or enhanced by introducing the SAP (whether dry, in asemi-solid gel, or dissolved in a liquid solution that is substantiallyfree of ions) into a fluid (e.g., a physiological fluid such as blood orgastric juice) or an area (e.g., by topical application on to thesurface of the eye, or by direct injection into the interior of the eye)including such ions. The gel does not have to be pre-formed prior toapplication to the desired site. Generally, self-assembly is expected tooccur upon contacting the SAP with such a solution in any manner.

A wide variety of ions, including anions and cations (whether divalent,monovalent, or trivalent), can be used. For example, one can promote aphase transition by exposure to monovalent cations such as Li⁺, Na⁺, K⁺,Cs+, and Ca⁺. The concentration of such ions required to induce orenhance self-assembly is typically at least 5 nM to 5 mM. Lowerconcentrations also facilitate assembly, although at a reduced rate.When desired, SAP can be delivered with a hydrophobic material (e.g.pharmaceutically-acceptable oil) in a concentration that permitsself-assembly, but at a reduced rate. When SAP are mixed with ahydrophobic agent such as an oil or lipid the assembly of the materialforms different structures. In some cases when another material isadded, the material will assemble into various other three-dimensionalstructures that may be suitable for loading of a therapeutic agent. Thehydrophilic portion of the molecule will assemble in such a way as tominimize hydrophobic-hydrophilic interaction, thereby creating a barrierbetween the two environments. Several experiments have shown that theSAP aligns on the surface of the oil with the hydrophobic portion of themolecule orienting toward the surface and the hydrophilic portion of themolecule facing away from the oil, or will form toroidal-like structureswith the hydrophobic material contained inside. This type of behaviorenables the encapsulation of therapeutics, prophylactic, or diagnosticmolecules of interest for delivery in the body.

The composition may contain a salt scavenger to drive assembly to apreferred configuration. For example, circular dichroism (“CD”)experiments indicate that the assembly dynamics can be controlled usingsalt scavengers or salt enhancement to increase the formation ofβ-sheets, α-helices, or more random configurations. The compositions mayoptionally contain an indicator showing the configuration of theassembly (e.g., α-helix, β-sheet, lattice, etc.).

Alternatively, some of the described materials do not require ions toself-assemble but may self-assemble due to interactions with solvent,hydrophobic interactions, side chain interactions, and hydrogen bonding.

The materials can be formed within regularly or irregularly-shapedmolds, which may include the surface of the eye, or a cavity on thesurface of the eye, or a portion of the eye (e.g., a tear in the cornea,or de-epithelialized section of the cornea) or which may be an inertmaterial such as plastic or glass. The structures or scaffolds can bemade to conform to a predetermined shape or to have a predeterminedvolume. To form a structure with a predetermined shape or volume (e.g.,a desired geometry or dimension, including thin sheets or films), anaqueous solution of the material is placed in a pre-shaped casting mold,and the materials are induced to self-assemble by the addition of aplurality of ions. Alternately, the ions may be added to the solutionshortly before placing the solution into the mold, provided that care istaken to place the solution into the mold before substantial assemblyoccurs. Where the mold is a tissue (e.g., within the eye or surroundingthe eye, whether in situ or not), the addition of an ionic solution maynot be necessary. The resulting material characteristics, the timerequired for assembly, and the dimensions of the macroscopic structurethat forms are governed by the concentration and amount of solution thatis applied, the concentration of ions used to induce assembly of thestructure, and the dimensions of the casting apparatus. The assembledmaterial can achieve a gel-like or substantially solid form at roomtemperature, and heat may be applied to facilitate the molding (e.g.,one can heat a solution used in the molding process (e.g., aprecursor-containing solution) to a temperature ranging up to about bodytemperature (approximately 37° C.)). Once the assembled material hasreached the desired degree of firmness, it can be removed from the moldand used for a described purpose. Alternatively, the materials describedmay be used to anchor host tissue to a tissue matrix or scaffold. Forexample, the described materials can be used as a “glue” to anchor hosttissue that is to be regenerated to a tissue matrix or scaffold toensure that the matrix or scaffold stays in place in the localenvironment to which it is injected or implanted. Tissue matrices andscaffolds are well known in the art and can be prepared from synthetic,semi-synthetic, and/or natural materials.

Materials that assemble and/or undergo a phase transition (e.g., atransition from a liquid state to a semi-solid, gel, etc.) when theycome in contact with bodily fluids (e.g., the tear film) or an ionicsolution are useful in providing an SAP structure at the surface or, orwithin one or more cavities of the eye. The SAP structure is effectiveto treat and prevent one or more symptoms of an eye disease. It may bethat the SAP reduce or prevent inflammation, reduce or prevent theformation of adhesions between diseased tissue and surrounding tissues,or induce and enhance the restoration of a damaged corneal epithelium.

Self-assembly or phase transition is triggered by components found in asubject's body (e.g., ions) or by physiological pH and is assisted byphysiological temperatures. Self-assembly or phase transition can beginwhen the compositions are exposed to or brought into contact with asubject's body (e.g., at the surface of the eye) and may be facilitatedby the local application of heat to the area where the composition hasbeen (or will be) deposited. Based on studies to date, self-assemblyoccurs rapidly upon contact with bodily fluids without the applicationof additional heat. The time required for effective assembly and/orphase transition can occur in 60 seconds or less (e.g., in 50, 40, 30,20, or 10 seconds or less) following contact with a subject's tissue, orto conditions similar to those found within the body. For example,solutions containing SAP can form a self-assembled fluid-impermeablestructure upon contact with physiological fluids within times as shortas 10 seconds following application. In some circumstances, such as whenconditions are sub-optimal (i.e., non-physiological), or when theconcentration of self-assembling precursors is low, self-assembly orphase transition may take longer to achieve, for example, up to aminute, 5 minutes, 10 minutes, 30 minutes, an hour, or longer.

The compositions can form structures that are substantially rigid (e.g.,solid or nearly solid) or that assume a definite shape and volume (e.g.,structures that conform to the shape and volume of the location to whicha liquid composition was administered, whether in vivo or ex vivo). Thesolidified SAP may be somewhat deformable or compressible after assemblyor phase transition, but it will not substantially flow from one area toanother, as compositions at a different point along the liquid to solidcontinuum may do, which may be due, at least in part, to their abilityto undergo phase transitions. As a result, the compositions can also beused to prevent the movement of a bodily substance in a subject in needthereof. Self-assembly can be achieved in vivo or ex vivo by exposure toconditions within a certain range of physiological values (e.g.,conditions consistent with the tears at the surface of the eye) ornon-physiological conditions. “Non-physiological conditions” refers toconditions within the body or at a particular site that deviate fromnormal physiological conditions at that site. Such conditions may resultfrom trauma, surgery, injury, infection, or a disease, disorder, orcondition. The SAP should self-assemble under such conditions. Whileliquid formulations are readily dispensed, the compositions administeredmay also be in a gel form that may become stiffer upon contact with thesurface of the subject's eye.

Regardless of the precise nature of the SAPs, upon exposure toconditions such as those described, the SAP can form membranous two- orthree-dimensional structures including a stable macroscopic porousmatrix having ordered or non-ordered interwoven nanofibers (e.g., fibersapproximately 5-20 nm in diameter, with a pore size of about 50-100 nmin a linear dimension). Three-dimensional macroscopic matrices can havedimensions large enough to be visible under low magnification (e.g.,about 10-fold or less), and the membranous structures can be visible tothe naked eye. Although three-dimensional, the structures can beexceedingly thin, including a limited number of layers of molecules(e.g., 2, 3, or more layers of molecules). Typically, each dimension ofa given structure will be at least 10 μm in size (e.g., two dimensionsof at least 100-1000 μm in size (e.g., 1-10 mm, 10-100 mm, or more)).The relevant dimensions may be expressed as length, width, depth,breadth, height, radius, diameter, or circumference in the case ofstructures that have a substantially regular shape (e.g., where thestructure is a sphere, cylinder, cube, or the like) or an approximationof any of the foregoing where the structures do not have a regularshape.

The SAP can form a hydrated material when contacted with water underconditions such as those described (e.g., in the presence of asufficient concentration (e.g., physiological concentrations) of ions(e.g., monovalent cations)). These may have a high water content (e.g.,approximately 95% or more (e.g., approximately 96%, 97%, 98%, 99% ormore)), and the compositions can be hydrated but not substantiallyself-assembled. A given value may be “approximate” in recognition of thefact that measurements can vary depending, for example, on thecircumstances under which they are made and the skill of the persontaking the measurement. Generally, a first value is approximately equalto a second when the first falls within 10% of the second (whethergreater than or less than) unless it is otherwise clear from the contextthat a value is not approximate or where, for example, such value wouldexceed 100% of a possible value.

The properties and mechanical strength of the structures or scaffoldscan be controlled as required through manipulation of the componentstherein. For example, the stiffness of an assembled gel can be increasedby increasing the concentration of SAP therein, as discussed above.Alternatively, it may be desirable for different parts of the SAPformulation to have different mechanical properties. For example, it maybe advantageous to alter the stability or density of the SAP formulationby manipulating the amino acid sequence. This may be desirable when theSAP formulations are used to fill a void, such that the edges of thematerial self-assemble to attach to the tissue site while the rest ofthe SAP formulation flows out into the void. In another example, thedensity of the material may be adjusted or sufficiently raised such thatit maintains enhanced resting contact with a healing retina, therebyallowing the patient greater freedom of head movement of the head. Thesequences, characteristics, and properties of the SAP formulations andthe structures formed by them upon self-assembly are discussed furtherbelow.

E. Additional Agents

The compositions of SAP can include other agents, such as therapeutic,prophylactic or diagnostic agents. The additional agents are typicallynon-self-assembling. These can be a biomolecule which is a molecule suchas a peptide, proteoglycan, lipid, carbohydrate, or a small molecule.Like small molecules, biomolecules can be naturally occurring or may beartificial (i.e., they may be molecules that have not been found innature). For example, a protein having a sequence that has not beenfound in nature (e.g., one that does not occur in a publicly availabledatabase of sequences) or that has a known sequence modified in anunnatural way by a human hand (e.g., a sequence modified by altering apost-translational process such as glycosylation) is an artificialbiomolecule. Nucleic acid molecules encoding such proteins (e.g., anoligonucleotide, optionally contained within an expression vector) arealso biomolecules and can be incorporated into the compositionsdescribed. For example, a composition can include a plurality of SAP andcells that express, or that are engineered to express, a proteinbiomolecule (by virtue of containing a nucleic acid sequence thatencodes the protein biomolecule).

Many different therapeutic, prophylactic or diagnostic agents can beincorporated into the formulation. One or more therapeutic, diagnosticand/or prophylactic agents can be administered simultaneously with theSAP in the same formulation, administered simultaneously in separateformulations, or sequentially. Alternatively, the agent(s) can becovalently or non-covalently coupled to the SAP, either directly or viaan intermediate molecule.

In some embodiments, compositions of SAP include one or morenon-self-assembling therapeutic agents. For example, the additionalagents can include one or more classes of therapeutic agents for thetreatment of ocular diseases, such as anti-inflammatory agents,vasoactive agents, anti-infective agents, anesthetics, growth factors,vitamins, nutrients, and/or cells. Additional therapeutic agents can beselected according to the disease that is to be treated, and the routeof administration. In some embodiments, additional therapeutic agentsare suitable for topical application to the eye. In other embodiments,additional therapeutic agents are suitable for parenteral application tothe eye.

In some embodiments, the compositions include one or more ophthalmicdrugs, including but not limited to anti-glaucoma agents,anti-angiogenesis agents, anti-infective agents, anti-inflammatoryagents, analgesics, anesthetics, growth factors, immunosuppressantagents, anti-allergic agents, anti-oxidants, cytokines, and combinationsthereof.

In some embodiments, the ophthalmic drug is present in its neutral form,or in the form of a pharmaceutically acceptable salt. In some cases, itmay be desirable to prepare a formulation containing a salt of an agentdue to one or more of the salt's advantageous physical properties, suchas enhanced stability or a desirable solubility or dissolution profile.Generally, pharmaceutically acceptable salts can be prepared by reactionof the free acid or base forms of an agent with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, non-aqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred.Pharmaceutically acceptable salts include salts of an agent derived frominorganic acids, organic acids, alkali metal salts, and alkaline earthmetal salts as well as salts formed by reaction of the drug with asuitable organic ligand (e.g., quaternary ammonium salts). Lists ofsuitable salts are found, for example, in Remington's PharmaceuticalSciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000,p. 704. Examples of ophthalmic drugs sometimes administered in the formof a pharmaceutically acceptable salt include timolol maleate,brimonidine tartrate, and sodium diclofenac.

In some embodiments, compositions of SAP include one or moreanti-glaucoma agents for local administration to the eye or surroundingtissues. Representative anti-glaucoma agents include prostaglandinanalogs (such as travoprost, bimatoprost, and latanoprost),beta-adrenergic receptor antagonists (such as timolol, betaxolol,levobetaxolol, and carteolol), alpha-2 adrenergic receptor agonists(such as brimonidine and apraclonidine), carbonic anhydrase inhibitors(such as brinzolamide, acetazolamide, and dorzolamide), miotic orparasympathomimetic drugs (such as pilocarpine and ecothiopate),serotonergic agents, muscarinic agents, dopaminergic agents, andadrenergic agents (such as apraclonidine and brimonidine).

In some embodiments, compositions of SAP include one or moreanti-angiogenesis agents for local administration to the eye orsurrounding tissues.

Representative anti-angiogenesis agents include, but are not limited to,antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®) and rhuFab V2 (ranibizumab, LUCENTIS®), and otheranti-VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanibsodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigmentepithelium derived factor(s) (PEDF); COX-2 inhibitors such as celecoxib(CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin-12(IL-12); thalidomide (THALOMID®) and derivatives thereof such aslenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozymeinhibitors such as ANGIOZYME® (Sirna Therapeutics); multifunctionalantiangiogenic agents such as NEOVASTAT® (AE-941) (AEterna Laboratories,Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors such assunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib(Nexavar®) and erlotinib (Tarceva®); antibodies to the epidermal grownfactor receptor such as panitumumab (VECTIBIX®) and cetuximab(ERBITUX®), as well as other anti-angiogenesis agents known in the art.

In some embodiments, compositions of SAP include one or morevasoconstrictor agents for local administration to the eye orsurrounding tissues.

Representative vasoconstrictors include epinephrine and phenylephrine.Vasoconstrictors such as phenylephrine can be included to prolong theeffect of local anesthesia (e.g., 0.1-0.5% phenylephrine). Analgesicagents other than a local anesthetic agent, such as steroids,non-steroidal anti-inflammatory agents like indomethacin, plateletactivating factor (PAF) inhibitors such as lexipafant, CV 3988, and/orPAF receptor inhibitors such as SRI 63-441.

In some embodiments, compositions of SAP include one or more localanesthetics for local administration to the eye or surrounding tissues.A local anesthetic is a substance that causes reversible localanesthesia and has the effect of loss of the sensation of pain. The SAPcomposition may include an anesthetic agent in an amount of, e.g., about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8% about 0.9%, about 1.0%, about 2.0%, about 3.0%, about4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, orabout 10% by weight of the total composition. The concentration of localanesthetics in the compositions can be therapeutically effective meaningthe concentration is adequate to provide a therapeutic benefit withoutinflicting harm to the patient.

In some embodiments, compositions of SAP include one or moreanti-infective or antimicrobial agents (e.g., an antibiotic,antibacterial, antiviral, or antifungal agent) for local administrationto the eye or surrounding tissues.

Other therapeutic agents can be included in the compositions, such asgrowth factors to accelerate one or more aspects of the recovery from,or prevention of a disease or disorder (e.g., angiogenesis, cellmigration, process extension, and cell proliferation). The growth factoror another agent can be a chemotactic substance, which has the ability,in vivo or in cell culture, to recruit cells to a site at which thesubstance is present. The cells recruited may have the potential tocontribute to the formation of new tissue or to augment and reinfoRCESexisting, damaged tissue (e.g., by contributing structurally and/orfunctionally to the tissue (e.g., by providing growth factors orcontributing to a desirable immune response)).

In some embodiments, the compositions include one or moreanti-inflammatory agents include steroid and non-steroid drugs. Suitablesteroids agents include glucocorticoids, progestins, mineralocorticoids,and corticosteroids. Small molecule steroidal anti-inflammatoriesinclude prednisone, dexamethasone, cortisone, loteprednol, triamcinoloneacetonide, fluocinolone acetonide, fluorometholone, and fluticasone.Exemplary immune-modulating drugs include cyclosporine, tacrolimus andrapamycin. In some embodiments, anti-inflammatory agents are biologicdrugs that block the action of one or more immune cell types such as Tcells, or block proteins in the immune system, such as tumor necrosisfactor-alpha (TNF-alpha), interleukin 17-A, interleukins 12 and 23.

The compositions can include a coloring agent. Suitable coloring agentsinclude commercially available food colorings, natural and syntheticdyes, and fluorescent molecules.

In some embodiments, the compositions include cells. Where cells aredelivered to the eyes of a patient (e.g., to treat or prevent one ormore diseases of the eye), autologous cells can be used.

F. Excipients, Carriers, and Devices

Compositions of SAP can include excipients suitable for administrationonto or into the eye. For example, compositions of SAP can be formulatedinto a composition suitable for topical administration onto the surfaceof the eye, or for injection into one or more of the cavities within theeye (see, for example, the schematic depiction of the eye in FIG. 1A).Formulations for application to the cornea are typically a liquidsolution or suspension. These may be injected into the eye, the tissuesurrounding the eye, or into a compartment of the eye, or topicallyapplied to the eye or cornea. Topical administration can includeapplication directly to exposed tissue, vasculature or to tissues orprostheses, for example, during surgery, or by direct administration tothe skin.

In the preferred embodiment, the formulation is a liquid orreconstitutable powder, applied topically. The formulations can includea pharmaceutically acceptable carrier or are provided as part of amedical device or coating.

In some forms, the formulation is provided as a dry or lyophilizedpowder which can be administered directly as a powder which hydrates atthe site of application.

Alternatively, the formulation is suspended or dissolved in a solvent,most preferably aqueous, and applied as a spray, paint, or injection.The formulation can also by administered in a hydrogel such as chitin,collagen, alginate, or a synthetic polymer. Any formulation suitable forapplication to the eye (e.g., a liquid, which can be applied as a sprayor a powder) can be used. In another embodiment, the formulation isprovided as a coating on a device, for example a contact lens oradhesive bandage, which may be dissolved in an aqueous solution anddried on the device or mixed with a polymeric carrier and applied to thedevice. In yet another embodiment, the formulation is provided in abandage, foam or matrix, in which the peptides may be dispersed orabsorbed.

The formulation can also be in the form of sutures, tape, or adhesive.In some embodiments, for example, where the formulation is administeredto the eye of a patient that has a disease or disorder relating to aprior injury to the eye, the SAP are formulated either alone, ortogether with other agents (e.g., with anesthetics, anti-inflammatories,growth factors, anti-infectives, etc.), in the form of a foam, matrix orbandage, for example to reduce or stop suppuration, bleeding or loss ofother bodily fluids, as required.

Suitable excipients can be selected based upon the desiredassembly-state of the self-assembling precursor materials. For example,when the SAP are delivered as a solution, a suitable excipient maycontain a concentration of ions below the threshold required to initiateassembly. Representative excipients include solvents, diluents, pHmodifying agents, preservatives, antioxidants, suspending agents,wetting agents, viscosity modifiers, tonicity agents, stabilizingagents, and combinations thereof. A preferred excipient is water.

Solutions, suspensions, or emulsions for ocular or intraocularadministration may be buffered with an effective amount of buffernecessary to maintain a pH suitable for ocular administration. Suitablebuffers are well known by those skilled in the art and some examples ofuseful buffers are acetate, borate, carbonate, citrate, and phosphatebuffers. Solutions, suspensions, or emulsions for ocular administrationmay also contain one or more tonicity agents to adjust the isotonicrange of the formulation. Suitable tonicity agents are well known in theart and include, for example, glycerin, mannitol, sorbitol, sodiumchloride, and other electrolytes.

In some instances, the formulation is distributed or packaged in aliquid form. Alternatively, formulations for ocular administration canbe packed as a solid, obtained, for example by lyophilization of asuitable liquid formulation. The solid can be reconstituted with anappropriate carrier or diluent prior to administration or applicationonto the eye.

Typically, when the composition is dispensed in a multi-dose containerthat is to be used over a longer period of time, such as 24 hours, apreservative must be added to ensure microbiologic safety over theperiod of use.

Numerous ophthalmological excipients are known in the art and available.They may contain suitable additives, such as preservatives,antioxidants, and stabilizing agents.

In some embodiments, compositions of SAP are formulated as an ophthalmicsolution for administration directly into the surface of the eye as aneye drop. Eye drops can contain SAP in any form suitable foradministration onto the surface of the eye, such as solutions,emulsions, suspensions and ointments.

In some embodiments, compositions of SAP are formulated as an ophthalmicemulsion. Ophthalmic emulsions are generally dispersions of oilydroplets in an aqueous phase. There should be no evidence of breaking orcoalescence.

In some embodiments, compositions of SAP are formulated as an ophthalmicsuspension. Ophthalmic suspensions contain solid particles dispersed ina liquid vehicle; they must be homogeneous when shaken gently and remainsufficiently dispersed to enable the correct dose to be removed from thecontainer. Sediment that may occur should disperse readily when thecontainer is shaken, and the size of the dispersed particles should becontrolled. The SAP precursors, or self-assembled SAP and any othersuspended material must be reduced to a particle size small enough toprevent irritation and damage to the cornea.

In some embodiments, compositions of SAP are formulated as an ophthalmicointment. Ophthalmic ointments are sterile, homogeneous, semi-solidpreparations intended for application to the conjunctiva or the eyelids.They are usually prepared from non-aqueous bases, e.g., soft paraffin(VASELINE®), liquid paraffin, and wool fat.

Ideally, the pH of ophthalmic drops should be equivalent to that of tearfluid, which is 7.4. However, the decision to add a buffering agentshould be based on stability considerations, as well as the conditionsrequired to maintain the SAP in the desired state of assembly. The pHselected should be optimized for both stability of the SAP andphysiological tolerance. For example, any buffer system must not causeprecipitation or deterioration of the SAPs. The influence on thelachrymal flow should also be taken into account. Although solutionswith the same pH as lacrimal fluid (7.4) are ideal, the outer surfacesof the eye tolerate a larger range, 3.5 to 8.5. The normal useful rangeto prevent corneal damage is 6.5 to 8.5. The final pH of the solution isoften a compromise, because many ophthalmic drugs have limitedsolubility and stability at the desired pH of 7.4. Exemplary pH valuesfor buffers formulated with excipients for topical administrationinclude pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5.

Ophthalmic solutions are ordinarily buffered at the pH of maximumstability of the drug(s) they contain. The buffers are included tominimize any change in pH during the storage life of the composition;this can result from absorbed carbon dioxide from the air or fromhydroxyl ions from a glass container. Changes in pH can affect thesolubility and stability of drugs; consequently, it is important tominimize fluctuations in pH. Therefore, in some embodiments, buffers orpH adjusting agents or vehicles are added to adjust and stabilize the pHat a desired level.

The buffer system should be sufficient to maintain the pH throughout theexpected life of the product, but with a low buffer capacity so thatwhen the ophthalmic solution is instilled into the eye, the buffersystem of the tears will rapidly bring the pH of the solution back tothat of the tears. In some embodiments, low concentrations of buffersalts (i.e., less than 20 mM, for example, 10 mM, or 5 mM) are used toprepare buffers of low buffer capacity that do not induce or otherwiseinfluence the self-assembly of the SAP or self-assemblingpeptidomimetics.

Ophthalmic drops are considered isotonic when the tonicity is equal tothat of a 0.9% solution of sodium chloride. The eye can usually toleratesolutions equivalent to 0.5-1.8% of sodium chloride. There are timeswhen hypertonic ophthalmic solutions are necessary therapeutically, orwhen the addition of an auxiliary agent required for reasons ofstability supersedes the need for isotonicity. A hypotonic ophthalmicsolution will require the addition of a substance (tonicity adjustingagent) to attain the proper tonicity range.

The most widely used ophthalmic buffer solutions are boric acid vehicleand Sorensen's modified phosphate buffer. The boric acid vehicle is a1.9% solution of boric acid in purified water or preferably sterilewater. It is isotonic with tears. It has a pH of approximately 5 and isuseful when extemporaneously compounding ophthalmic solutions of drugsthat are most stable at acid pH. This vehicle does not possess largebuffering capacity, but it is sufficient to stabilize pH for the shortexpiratory periods used for compounded solutions, without overwhelmingthe natural buffers in lacrimal fluid. The second most commonly usedbuffer solution is the Sorensen's modified phosphate buffer and is usedfor drugs needing pH values between the range of 6.5-8.0. This bufferuses two stock solutions, one acidic containing NaH₂PO₄, and one basiccontaining Na₂HPO₄. Preferred ophthalmological excipients for topicaladministration are biocompatible, non-toxic and do not induceinflammation.

For application by the ophthalmic mucous membrane route, compositionsmay be formulated as eye drops or eye ointments. Typically, the eyedrops are in the form of an aqueous solution, for example, including oneor more excipients suitable for administration into the eye. Mostformulations applied topically as drops are administered as volumes of0.03 and 0.07 ml/drop, with a maximum typical volume of about 100microliters. The formulations may also be provided in two units, onelyophilized and one a diluent for re-suspension of the lyophilizedagent(s).

In some forms, compositions of SAP are formulated to include apharmaceutically acceptable excipient for parenteral administration tothe eye. For example, injectable solutions can be prepared byincorporating the SAP in the required amount in the appropriate solventor dispersion medium with one or more pharmaceutically acceptableexcipients, as required. Generally, dispersions are prepared byincorporating the various compositions into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredients.In the case of powders for the preparation of injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the SAP plus any additional desiredingredient from a previously prepared solution thereof.

In some forms, pharmaceutical formulations for administration byinjection are an aqueous solution or suspension of the SAPs. Inpreferred embodiments, formulations for injection into the eye includeless than 20 mM ions, for example, between 20 mM and 0.01 mM ions,inclusive. In a particular embodiment, formulations of SAP for injectioninto the eye include a solution of the SAP in water. In otherembodiments, the solutions of the SAP include one or more polymerconjugates. Exemplary solvents include, for example, water, Ringer'ssolution, phosphate buffered saline (PBS), and isotonic sodium chloridesolution. The formulation may also be a sterile solution, suspension, oremulsion in a nontoxic, acceptable diluent or solvent such as1,3-butanediol.

Solutions, suspensions, or emulsions for injection or instillation intothe eye may be combined with an effective amount of buffer necessary tomaintain a pH suitable for ocular administration. Suitable buffers arewell known in the art, Examples include acetate, borate, carbonate,citrate, and phosphate buffers.

Solutions, suspensions, or emulsions for injection or instillationadministration may contain at least one tonicity agents to adjust theisotonic range of the formulation. Suitable tonicity agents are wellknown in the art. Examples include glycerin, mannitol, sorbitol, sodiumchloride, and electrolytes.

Formulations for intravitreal injection can be formulated to a desiredvolume. For example, the volume suitable for intravitreal injection istypically between about 0.03 and 0.1 ml. Preferred excipients forintravitreal injection are biocompatible, non-toxic and do not induceinflammation.

Solutions, suspensions, or emulsions for ocular administration maycontain one or more preservatives to prevent bacterial contamination ofthe ophthalmic preparations. Suitable preservatives known in the artinclude polyhexamethylene biguanide (PHMB), benzalkonium chloride (BAK),stabilized oxychloro complexes (otherwise known as Purite®),phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine,benzyl alcohol, parabens, thimerosal, and mixtures thereof.

Solutions, suspensions, or emulsions for ocular administration may alsocontain one or more excipients known in the art, such as dispersingagents, wetting agents, and suspending agents.

The compositions of SAP can include additional organic and/or inorganicmaterials, for example, to provide a structure or physical support forthe SAP. In some embodiments, the additional materials providestructural support to the compositions, such as materials that provide ascaffold. Scaffold materials can be selected to provide physicalstrength, elasticity, porosity, solubility, volume and bulk, as requiredby the application. In certain embodiments, the scaffold material hasmechanical and/or biological properties similar to that of theextracellular matrix (ECM).

Scaffold materials can include natural or synthetic polymers, includingnatural polymers such as polypeptides and proteins, may create ascaffold onto which SAP, therapeutic agents, cells or other agents areattached or associated. In some embodiments, the described compositionsinclude proteins, such as ECM proteins. Exemplary natural scaffoldmaterials include alginate, fibrinogen, hyaluronic acid, starch,chitosan, silk, gelatin, dextran, elastin, collagen, and combinationsthereof. In some embodiments, compositions of SAP include scaffoldmaterials that are synthetic polymers. Exemplary synthetic polymersinclude poly(L-lactic acid co-ε-caprolactone) (PLCL), polyhydroxy acidssuch as poly(DL-lactic acid) (PDLA) and poly(lactic-co-glycolic acid)(PLGA), poly(ethylene oxide) (PEO); poly(vinyl alcohol) (PVA; poly(methyl methacrylate) (PMMA), poly(ethylene-co-vinyl acetate) (PEVA),polystyrene; polyurethane; and mixtures thereof. In preferredembodiments the scaffold materials are biocompatible. In preferredembodiments the scaffold materials do not induce an immune response.

SAP structures can biodegrade at a time following application that isconsistent with the time required for treatment, for example, the amountof time required for restoration or regeneration of the cornealepithelium, such as one day, one week, one month or more than one monthfollowing application.

Contact lenses including SAP can be used daily, overnight or long-term.Contact lenses including SAP can be formulated for use in therapeutic orcosmetic applications, according to the needs of the intended recipient.In some embodiments, SAP are applied to commercially available contactlenses, such as commercially-available cosmetic or therapeutic contactlenses. Application can occur before or after assembly of the SAP hasoccurred, and can be carried out by any suitable means known in the artfor application, such as spraying, coating, painting, etc. Contactlenses including SAP can include one or more therapeutic, diagnostic orprophylactic agents.

When the contact lens does not contain an additional scaffold or supportstructure, additional agents can be mixed into the SAP prior to assemblyto form an SAP structure having non-self-assembling agents incorporatedor entrapped therein, or bound directly to the SAP prior to assembly. Insome embodiments, additional agents are applied to one or more surfacesof an SAP structure, which is subsequently applied to the eye for use asa contact lens, or which is incorporated into one or more additionalstructures for use as a contact lens.

In some embodiments, contact lenses including SAP are optically clear.For example, the SAP and any support structures or materials areoptically clear or transparent when desired. In other embodiments, SAPfor use in contact lenses are formulated to include one or more coloringagents or dyes to, for example, prevent, reduce or otherwise alter thepassage of light through the contact lens. For example, in certainembodiments, contact lenses including SAP are dyed or tinted to reduceor limit exposure of light to the eye. When support structures orsurfaces are used, the support structure or surface can be colored ordyed to alter the amount or wavelength of light that reaches the eye.The SAP can include a coloring agent or dye in an amount sufficient tocompletely obscure the passage of light through the assembled structure.

Contact lenses can include a single SAP structure or can include two ormore distinct layers of SAP structures, for example, including the sameor different SAP and optionally including one or more additional agents.

Contact lenses can be packaged and stored in appropriate containers. Thelenses can be stored within a suitable fluid, for example, contact lenssolution. In some embodiments, the storage fluid or contact lenssolution includes one or more SAPs. When contact lens solution includesone or more SAP, the ionic concentration of the solution is typicallybelow 20 mM. The SAP can be added to the solution as required, forexample, to maintain a constant concentration. In some embodiments,contact lenses are continually worn and immersed within a solutioncontaining self-assembling precursor materials, for example, to maintainan SAP structure at the surface of the lens, for application to thesurface of the eye.

Ocular wound bandages including SAP are described. Ocular wound bandagescontaining SAP can be formulated for use in therapeutic or cosmeticapplications, according to the needs of the intended recipient. In someembodiments, SAP are applied to commercially available ocular woundbandages, such as commercially-available gauzes or eye patches.Application can occur before or after assembly of the SAP has occurred,and can be carried out by any suitable means known in the art forapplication, such as spraying, coating, painting, etc.

In certain embodiments, compositions of SAP are applied in the form of asolution or powder directly onto gauze or other non-peptide structures.For example, SAP and/or scaffold materials can be contacted with tissuearound and within the eye, and held in place by a bandage or gauze, oras one component of an eye patch.

G. Kits

The SAP can be assembled in kits, together with instructions for use.The kit may also include one or more of a syringe (e.g., a barrel orbulb syringe) a dropper or dropper bottle, a needle, a pipette, gauze,sponges, or cotton, swabs, an eye bath, a bandage, a contact lens, aneye patch, a disinfectant, surgical thread, scissors, a scalpel, asterile fluid, a spray canister, including those in which a liquidsolution is sprayed through a simple hand pump, a sterile container,disposable gloves or an eye dropper.

Exemplary kits include SAP, self-assembling peptidomimetics, orcombinations of SAP and peptidomimetics, excipients, suitable means forapplication, and instruction for use. In some embodiments, kits includemeasured dosages of one or more SAP and/or self-assemblingpeptidomimetics for application at distinct times. For example, a kitcan include SAP having a different sequence, dose, or conjugated to adifferent or the same agent(s) for distinct applications into the sameeye. Dosages, and application regimens can be determined according tothe needs of the patient, for example, as specified by a physician orwithin the instructions.

Kits can include one or more means for administration into or onto theeye. Typically, the applicator will initiate or maintain contact betweenone or more SAP with one or more parts of the eye. In some embodiments,the applicator is pre-filled or loaded with the SAP. When SAP containedwithin the applicator, the amount and formulation of the SAP formulationto be dispensed can be fixed or varied, for example, by the patient orphysician. Exemplary, applicators include an eye-dropper, a syringe, aneye bath, a spray, an air-applicator, a contact lens, an eye patch and atube. In some embodiments, the SAP are provided in one or morecontainers, for example, in dried form (e.g., lyophilized), or asolution, tablet, wafer, or gel. In some embodiments, the SAP arepre-packaged in a concentration stock powder or solution, withinstructions for diluting to the desired concentration prior toapplication.

Kits including dried SAP can be packaged with a desiccant. In someembodiments, kits include one or more pharmaceutically acceptableexcipients for administration into the eye. The excipient can becontained within a separate container or within an applicator.Therefore, in some embodiments, an applicator can include one or moreSAPs, and one or more excipients within the same or differentcompartments. In some embodiments, one or more SAP is diluted orotherwise mixed with one or more excipients within the applicator priorto application. The amount and type of SAP to be administered can bepre-determined within the applicator or varied according to the desiredeffects.

In some embodiments, kits include an air applicator for the directedapplication of SAP in the form of a powder to the surface of the eye.The amount of SAP administered can be varied. The contacted surface areaof the eye can be adjusted, for example, to a narrowly-defined area orthe entire exposed portion of the eye. In other embodiments, kitsinclude an apparatus to deliver a powder to the eye via a ballisticinjection through the outer layers of the eye. In other embodiments,kits include an apparatus to deliver a solution or gel via a single orseries of injections or infusion.

In some embodiments, kits include an applicator that will mix two ormore different SAP together, to be dispensed and/or delivered togetherin one application or in a series of applications. For example, thedevice can contain several chambers, each of which contains an SAP whichis specific for the eye. The composition can be dispensed directly ontothe site of administration or can be mixed in a mixing chamber withinthe device prior to administration. In one embodiment, an applicator canbe used to administer the same composition to the eye on severaldifferent occasions or different compositions to the eye at the same ordifferent times.

III. Methods of Making SAP and Compositions Thereof

Compositions of SAP can be prepared using any techniques known in theart. SAP are typically synthesized using standard procedures, so anytechnique in the art suitable to prepare synthetic peptides can be used.All of the described method steps can be performed in any suitable orderunless otherwise indicated or otherwise clearly contradicted by context.

A. Production of SAP

SAP can be chemically synthesized or purified from natural orrecombinantly-produced sources, by methods well known in the art. Forexample, peptides can be synthesized using standard Fmoc chemistry.

Standard Fmoc (9-florenylmethoxycarbonyl) derivatives includeFmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Ala-OH. Couplings aremediated with DIC (diisopropylcarbodiimide)/6-Cl-HOBT(6-chloro-1-hydroxybenzotriazole). In some embodiments, the last fourresidues of the peptide require one or more recoupling procedures. Inparticular, the final Fmoc-Arg(Pbf)-OH coupling can require recoupling.For example, a second or third recoupling can be carried out to completethe peptide using stronger activation chemistry such as DIC/HOAT(1-hydroxy-7-azabenzotriazole) or HATU(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate)/NMM (N-methylmorpholine).

Acidolytic cleavage of the peptide can be carried out with the use ofcarbocation scavengers (thioanisole, anisole and H₂O). Optimization canbe achieved by varying the ratio of the components of the cleavagemixture. An exemplary cleavage mixture ratio is 90:2.5:2.5:5(TFA-thioanisole-anisole-H₂O). The reaction can be carried out for 4hours at room temperature.

In some embodiments, the removal of residual impurities is carried outby wash steps. For example, trifluoroacetic acid (TFA) and organicimpurities can be eliminated by precipitation and repeated washes withcold diethyl ether and methyl t-butyl ether (MTBE).

Peptides produced using the disclosed methods can be purified using highpressure liquid chromatography (HPLC). Suitable solvents for dissolvingthe peptides include neat TFA. In some embodiments, 8 mL TFA/g peptideis sufficient to fully dissolve peptides following precipitation. Forexample, TFA can be diluted into H₂O. Typically, the peptides remainsoluble at TFA concentrations of 0.5% to 8% and can be loaded ontoreverse phase (RP)-HPLC columns for salt exchange. Exemplary saltexchange methods use 3-4 column volumes of acidic buffer to wash awaythe TFA counter ion due to its stronger acidity coefficient. Bufferssuitable for use in washing away the TFA counter ion include 0.1% HCl inH₂O.

Following removal of TFA, peptides can be eluted with a step gradient.Exemplary elution buffers include 30% acetonitrile (MeCN) vs. 0.1% HClin H₂O. For acetate exchange, peptides can be loaded from the samediluted TFA solution, washed with 3-4 column volumes of 1% acetic acid(AcOH) in H₂O, followed by 2 column volumes of 0.1 M NH₄OAc in H₂O, pH4.4. In some embodiments, the column is washed again with 3-4 columnvolumes of 1% AcOH in H₂O.

Peptides can be eluted from the columns using a step gradient of 30%MeCN vs. 1% AcOH in H₂O. In some embodiments, the elution of peptidescan be enhanced by acetate exchange. Exemplary buffers for acetateexchange include 0.1 M NH₄OAc in H₂O, pH 4.4.

Analytical HPLC can be carried out to assess the purity and homogeneityof peptides. An exemplary HPLC column for use in analytical HPLC is aPHENOMENEX® JUPITER® column. In some embodiments, analytical HPLC iscarried out using a column and buffer that are heated to a temperature sgreater than 25° C., for example 25-75° C. In a particular embodimentanalytical HPLC is carried out at temperatures of about 65° C. A stepgradient can be used to separate the peptide composition. In someembodiments, the gradient is from 1%-40% MeCN vs 0.05% TFA in H₂O. Thechange in gradient can be achieved over 20 min using a flow rate of 1ml/min. Peptides can be detected using UV detection at 215 nm.

In some embodiments, methods of making the described compositions foradministration to the eye include the step of sterilization. Wherecompositions are required to be sterilized or otherwise processed forthe removal of undesirable contaminants and/or micro-organisms,filtration is a preferred method. Filtration can be achieved using anysystem or procedures known in the art. In some embodiments, filtrationremoves contaminants or prevents the growth or presence ofmicroorganisms. Exemplary microorganisms and contaminants that can beremoved include bacteria, cells, protozoa, viruses, fungi, andcombinations thereof. In some embodiments, the step of filtration iscarried out to remove aggregated or oligomerized proteins. For example,solutions of self-assembling precursor peptides or peptidomimetics canbe filtered to remove assembled peptide structures or oligomers on thebasis of size.

B. Fabrication of Compositions for Treating Eye Disease

When the SAP are used to form a gelled or solid structure, for example,to be applied to the eye as part of a backing on a contact lens, the SAPformulations can be produced using one more techniques. Examples includetemplating onto the surface; injection molding of a formulation of SAPin a solvent; stamping of a dry powder or frozen formulation of SAP in asolvent; direct application of a slurry formulation containing SAP ontoa stencil or patterned surface, such as a bandage, adhesive bandage or acomposite structure formed by a combination of these methods.

In some embodiments, SAP formulations are dried or dehydrated to removea solvent. Any methods known in the art can be used for the dehydrationof compositions including SAP.

The term “dried” or “lyophilized” is used to describe the product of aprocess for the removal of the majority of the solvent from a materialin solution, for example, by methods for dehydration, vacuum sublimationor “lyophilization”. These methods typically remove a major portion ofsolvent, such as water, from the material, but can result in a residualamount of solvent within the “dry” product. For example, a lyophilizedpowder may include up to 10% w/w of water, for example, 5%, 3%, 2%, 1%or 0.5% w/w of water.

Any composition or formula of SAP can include up to 25% by mass ofderivative molecules and/or degradation products. Exemplary derivativemolecules and/or degradation products include, but are not limited to,amino acid substitutions, amino acid deletions, oligomers includingdimer, trimmers, tetramers or higher-order oligomers, aggregates andimpurities. In some embodiments, formulations of SAP include less thanor equal to 20% derivative molecules and/or degradation products bymass, such as 15%, 10% or less than 10%, such as 5%, 1% or 0%.

IV. Methods of Use

A. Routes of Administration and Dosages

SAP can be used for the treatment and prevention of eye diseases.Methods of using SAP and compositions thereof for the treatment andprevention of eye diseases typically include administering the SAPdirectly to the eye, either topically or by injection or byinstillation.

Dosage units containing an amount of SAP to provide pain relief in theeye or ocular cavity are also provided. Dosage units can be prepared inan amount effective to prevent, reduce or inhibit sensory function atthe surface of the eye in an amount sufficient to reduce pain associatedwith one or more diseases, disorders, injuries and related symptoms thataffect the eye.

SAP used in the compositions to treat eye diseases can be assembledprior to or at the time of application of the composition to the eye,either by contacting the composition with an ionic solution or allowingthe composition to contact a bodily fluid.

In preferred embodiments, SAP are administered once, twice, three timesor more than three times a day directly to the eyes of the individual inneed thereof. The frequency will vary depending on the severity ofsymptoms. The formulation may be applied as a drop in the form of anemulsion or suspension, lotion, ointment, cream, gel, salve or powderand sustained or slow release, as well as eyelid lotion. It may also beused as an eye wash or rinse to irrigate the eye. The formulation mayalso be applied in a sprayable form.

In preferred embodiments, the dosage administered to the eye does notresult in toxicity or inflammation within or at the surface of the eye.For example, multiple applications per day for an extended period oftime do not result in damage or toxicity to the cornea.

Exemplary conditions that can be treated include DED, RCES, ocular painor irritation associated with a contact lens or ocular prosthesis.Methods for treating or preventing pain associated with eye surgery anddisorders or diseases of the eye are also provided. An exemplaryprosthetic or medical device that can be coated, treated or otherwiseassociated with SAP is an intraocular lens. Replacement lenses that areimplanted inside the eye to replace the eye's natural lens when it isremoved during cataract surgery are known in the art. In someembodiments, coating, covering or otherwise associating an intraocularlens with SAP prior to, during or immediately following cataract surgeryenhances the outcome of cataract surgery. For example, in someembodiments, implanting an intraocular lens coated with SAP into asubject reduces or prevents posterior capsule opacity (PCO) and/orintraocular lens dislocation relative to implanting an untreated controlintraocular lens. In some embodiments, the formulation is used in theimplantation of complicated retinal grafts (photoreceptors and pigmentepithelium) or in Pterygia/pterygium surgery, including use ofautologous conjunctival autografting.

Methods of administering SAP into or onto the eye to control (e.g.,prevent) or treat diseases, disorders, injuries and related symptomsthat affect the eye in subjects are described. In some embodiments, SAPcan be administered into or onto the eye either prophylactically or inresponse to disruption of the tear film or reduced tear production in asubject. For example, in some embodiments, the compositions areadministered to prevent, treat, or reduce the symptoms of a disease,disorder, injury or related. The compositions can be used prior to or atthe time of or after eye surgery or to restore and augment one or morelayers of the surface of injured, damaged or diseased cornea in asubject in need thereof. Damage to the cornea can result from traumaticinjury, or surgical incisions, or chemical or heat burns, or as a resultof an infectious agent, such as a viral, fungal, bacterial or protozoanpathogen, from local or systemic diseases and disorders, such asglaucoma or diabetes, or from chemical imbalances, contact lens use,disease or aging leading to disruption of the tear film. Disorders mayarise due to traumatic injury, surgery, burns, viral, fungal, bacterialor protozoan infection, acquired or congenital diseases and disorders ofthe eye or ocular cavity.

SAP can be used to repair, replace or augment one or more of thebiological structures of the surface of the eye, for example, byproviding an optically clear coating across the surface of the eye. Insome embodiments, the methods treat and augment one or more layerswithin the tear film at the surface of the cornea, including theexternal lipid layer, the aqueous layer, the mucus layer, the epitheliallayer, and the corneal stroma.

The SAP are administered in an amount sufficient to coat or cover thediseased or damage portion of the cornea. In some embodiments, theamount of SAP administered is sufficient to cover the entire exposedsurface of the cornea. For example, SAP are administered as a solutionin a volume sufficient to form a coating of up to 1 mm in thickness overthe entire exposed surface of the cornea. The SAP self-assemble uponcontacting the ocular surface to form a visually clear structure thatcovers the surface of the eye. The SAP structure can be of uniformthickness sufficient to augment and/or reproduce the outer surfacelayer(s) of the natural cornea and/or recreate the biological functionsof the outer surface layer(s) of the cornea. In other embodiments, theSAP are administered to the eye to reduce or mediate one or moreinflammatory responses associated with a disease or damage to one ormore structures of the eye.

In some embodiments, the methods can reduce, prevent or otherwisecontrol the biological functions of one or more cell types in orsurrounding the eye. An exemplary cell type is a corneal keratinocytecell. For example, in some embodiments, SAP are administered to the eyein an amount sufficient to prevent or reduce the deleterious effects ofcorneal scarring and opacity associated with uncontrolled activation ofcorneal keratinocytes in response to corneal damage. Therefore, methodsfor reducing the activation and/or proliferation of cornealkeratinocytes by administering SAP to the eye are also provided. In someembodiments, the methods reduce or prevent the onset of scarring andfibrosis following damage to one or more of the structures of the eye,for example, associated with disease, trauma, or surgery. Exemplarysurgical procedures of the eye include corneal transplant surgery,cataract removal/lens replacement surgery, pterygium removal surgery,conjunctival surgery, incisional glaucoma surgery, refractive surgerysuch as LASIK or PRK procedures, and other invasive eye procedures. Insome embodiments, the methods reduce or prevent the onset of scarringand opacity following damage to the corneal surface, for example,associated with corneal surgery. Therefore, methods of prophylacticallytreating a site of disease or damage of the eye surface to maintainvisual acuity or prevent or reduce the rate of deterioration of visualacuity are also provided. In some embodiments, SAP are administered toone or more structures of the eye to prevent the loss of vision, or torestore or enhance the vision of a subject in need thereof.

SAP can be administered via any means which is effective to deliver aneffective amount of the SAP to the eye. For example, compositions can beadministered by topical, intra-corneal, intravitreal, intracameral,periocular, punctual, subconjunctival, sub-tenon, subchoroidal,suprachoroidal and subretinal routes. Exemplary ocular compartments intowhich SAP can be administered include the vitreous chamber, subretinalspace, subchoroidal space, episclera, conjunctiva, sclera, anteriorchamber, and/or the cornea and compartments therein (e.g.,subepithelial, intrastromal, endothelial).

The SAP can be applied to the outer surface of the eye, tissuescontacting or surrounding the eye, or into one or more inner compartmentto be treated. Exemplary tissues that can be contacted with the SAPinclude the lids, lacrimal glands, lacrimal ducts, conjunctiva, sclera,cornea, aqueous humor, iris, ciliary body, trabecular meshwork, lens,vitreous humor, retina, choroid, optic nerve and adnexa.

The compositions can be administered as a liquid or powder containingsubstantially non-assembled SAP (i.e., SAP that has yet to undergo aphase transition from the liquid-to-solid state), or they can beadministered in the form of a solid (i.e., as a substantially assembledgel). In other embodiments, the SAP can be administered to the eye as anemulsion, for example, as a mixture of solid particles includingsubstantially-assembled peptides, and non-assembled precursor peptides.

In some embodiments, formulations of SAP are delivered directly to oneor more of intraocular cavities within the eye using a syringe or othermeans for puncturing the outer surface of the eye and delivering theSAP. An exemplary procedure for administering formulations to the eye isintravitreal injection. Intravitreal injection is an injection into thevitreous, which is the jelly-like substance inside the eye. It isperformed to place formulations inside the eye, such as near the retina.The formulations of SAP can be injected using a syringe, or othersuitable deliver means.

In some embodiments, formulations of SAP are delivered directly to thesurface of the eye using mechanical delivery means, i.e., dropping,spraying, bathing of the eyes in a solution, or a combination of these.Most formulations applied topically as drops are administered as volumesof 0.03 and 0.07 ml/drop, with a maximum typical volume of about 100microliters/drop.

In some embodiments, a powder of SAP is applied to the surface of theeye using an air applicator that can be adjusted for directedapplication to a narrow area or the entire exposed portion of the eye.In other embodiments, the powder can be delivered to the eye via aballistic injection through the outer layers of the eye. In anotherembodiment, an applicator can mix several SAP together to be deliveredin one or a series of applications or in a staged delivery system viawhich components or combinations thereof are delivered in a specificorder to achieve a desired structure based on the sequence of delivery.The sequence of delivery can result in different structures by varyingthe amount and the form of delivery. In one example, the deliverysequence can begin with a powder, which is then followed by a liquid,which is in turn followed by a powder to result in a triple layeredstructure that can conform to specific shapes based on the area andvolume covered.

The SAP can be assembled before, during or after application to theeyes. For example, the SAP can be synthesized and the finishedformulation exposed to an ionic solution to induce gel formation. Thegelled structure can be stored until use. The gelled structure can bedehydrated prior to storage. The structure can be gelled in a mold toform a particular shape, for example, to form a layer of on the surfaceof a soft contact lens. In other embodiments, the SAP precursors aresynthesized and stored in a substantially non-assembled form. Thenon-assembled SAP precursors can be dried prior to storage. Immediatelyprior to use, the dehydrated or dried non-assembled SAP can be exposedto an ionic solution to initiate assembly. The SAP can be gelled in amold to form a particular shape. In still other embodiments, the gelledstructure is applied or implanted in an unassembled form and thepeptides assemble upon contact with a bodily fluid, such as the tearfilm. This can be useful, for instance, to allow SAP to assemble andconform to the shape of the application site.

In some embodiments, methods include administering formulations of SAPincluding one or more therapeutic, prophylactic, and/or diagnosticagents, as discussed above. When the SAP are applied in the form of agelled structure, the agent can be impregnated into an SAP structureand/or coated on the surface of the structure. In other embodiments, themethods include administering an agent that is covalently coupled to oneor more of the SAPs. For example, in some embodiments, the formulationsof SAP include a pH-adjusting agent which is released at the site ofadministration to alter the pH at the site of administration.

Since the SAP form an optically clear SAP structure, the methods caninclude creating a self-assembled barrier-structure that acts as amolecular “exclusion zone” at a desired location within the eye, forexample, to prevent undesirable cellular proliferation. Typically, theformation of a self-assembled barrier that prevents passage of bodilyfluids through the structure requires an SAP concentration of greaterthan about 1% w/v, for example, and amount between 1% and 4% w/v,inclusive. In an exemplary embodiment, the methods provide aself-assembled barrier-structure over the foveal region of the retina toexclude the growth of blood vessels in this area. Typically, the methodspreserve normal vision throughout the healing process. The methods canbe used in place of, or in addition to existing therapeutic techniques,such as laser removal of the blood vessels that have grown over thefovea. The methods result in an enhanced outcome relative to existingmethods and protect the foveal region of the retina while preservingnormal vision.

In another embodiment, SAP can be used to fill the vitreous cavity inlieu of or in combination with sterile saline (salt water) or with avitreous substitute such as a gas bubble or silicone oil for posterioreye procedures, such as to lessen traction on the retina, to closeretinal tissue breaks and minimize bleeding in retinal repair, orprevent proliferative vitreoretinopathy (PVR) by providing a barrier tostabilize the structure and minimize retinal pigment epithelial cellabnormal migration and proliferation. Further, adequate density of SAPcould be valuable to lessen constraints on head movement and positionfor the recovering post-operative surgical patient.

In some embodiments, the SAP applied at the surface of the cornea canpass into and across the cornea to enter the inner compartments of theeye. For example, Cy5.5 conjugated to a RADARADARADARADA peptide(Cy5.5(RADA)₄CONH₂; SEQ ID NO: 434) administered onto the healthycorneal surface was shown to localize within the retina. In contrast,the Cy5.5 dye alone was not transported across the cornea. Specifically,a solution of Cy5.5 conjugated to RADA (Cy5.5(RADA)COOH; SEQ ID NO:442), Cy5.5 conjugated to RADARADA (Cy5.5(RADA)₂COOH; SEQ ID NO: 444),and Cy5.5 conjugated to (RADA)₄ (Cy5.5(RADA)₄CONH₂; SEQ ID NO: 434)administered as eye drops was shown to localize to the retina. It may bethat the size and/or sequence of the SAP is sufficient to enable passiveor active uptake of non-assembled peptide monomers by transportprocesses at the surface of the cornea. The size of the RADA-16 peptideis approximately 10 nm.

Therefore, SAP bound to one or more agents can transport bound agentsthrough the corneal surface without causing damage or inflammation ofthe cornea or the ocular compartments. The methods direct SAP across thecornea without the need for injections, penetration enhancers orsurgical implements that pierce the cornea and risk potential infectionin the eye.

In some embodiments, administration of the SAP to the corneal surface iscarried out in two or more consecutive applications, to produce two ormore distinct layers of SAP structures at the surface of the cornea.

The surface of the eye contains multiple stacked layers, including theoil (top) layer; the aqueous (second) layer; the mucous (third) layer,and the epithelial (base) layer (see FIG. 1B). The outer three layersform the tear film, which constantly changes, at least in part due toblinking. The structure and function of each layer can vary, dependingon the disease state of the eye. For example, in high pressure glaucoma,typically, tear duct outflow from the tear ducts is reduced, and thecorneal surface structure changes. The reduction of outflow can lessenthe overall volume of liquid available to the eye surface, and it cancause elongation, or “stretching” of the conformation of collagen fiberspresent within the cornea. The difference in the surface area of thecollagen can vary by as much as 15%, opening pores within the tissue,thereby enhancing the risk of infection and causing pain or discomfortin the eyes. Each blink of the eyelid (opening and closing) displacesapproximately 80% of the fluid content of the tear film, eye drop, orother fluid placed on the eye. To counteract this loss, SAP administeredto the eye needs to be able to interact with a low degree of mobility toadhere to the eye. Therefore, in some embodiments, administration of SAPonto the corneal surface provides a first SAP structure or “layer” thatis directly in contact with the surface of the cornea. In someembodiments, the SAP provided in the first administration includes oneor more tissue-specific motifs, for example, to bind to the cornealsurface. An exemplary tissue-specific motif is the sequence MSCRAMM (SEQID NO: 141).

A second administration of the SAP onto the corneal surface provides asecond SAP structure or “layer” that is directly in contact with thefirst layer. The second or other middle layer(s) can have a combinationof hydrophilic material to enable fluid flow and maintain the correctorientation of the third or outer layer. Therefore, the second caninteract with the first layer and be an intermediary to anchor thethird, or further layer(s).

A third administration of the SAP onto the corneal surface provides athird SAP structure or “layer” that is directly in contact with thesecond layer. In some embodiments the third layer is sensed when theeyelid moves over the eye. In some embodiments, the third layer can forma fluid-resistant barrier to reduce the evaporation due to theorientation of the hydrophobic layer to the air. The SAP for use in eachof the layers can be designed according to the needs of the subject, forexample, to reduce evaporation, to reduce or prevent bacterial, viral orfungal infection at the surface of the cornea, prevent dust and debrisfrom entering the tear film, etc. Therefore, each of the layers can bedesigned to provide relatively greater or reduced fluid flow between thelayers. In some embodiments, one or more of the layers is a barrier tofluid movement. In other embodiments, one or more of the layers directsthe movement of fluids out of the eye or the penetration of gases intothe eye.

To form layers at the surface of the eye, the SAP can be administered tothe corneal surface in consecutive applications, followed by a timeinterval sufficient to allow assembly of the SAP. An exemplary timeinterval is between about 30 seconds and 10 minutes, for example, 1, 2,3, 4, 5 or 6 minutes between applications. Different or equivalentadministration forms, for example, powders and solutions can be used forsuccessive applications, according to the needs of the patient and thedesired properties of the resulting structures. For example, applicationof a powder may provide an SAP structure having greater interaction withthe ocular surface than does a solution.

In some embodiments, SAP is administered to the eye to produce a depotin or around the eye. In an exemplary embodiment, the SAP are introducedwithin a slow-release formulation into the eyelid, for example, whenimplanted within a medical device. In order to counteract fluid loss dueto blinking, for example, SAP can be slowly and continuously releasedacross the surface of the eye following each blink.

In an exemplary embodiment, SAP are applied to the eye with, or as partof, such as a coating on, a medical device, for example, as a solutionfor slow-release from the medical device. In an exemplary embodiment, amedical device for dispensing the SAP is embedded within one or morestructures proximal to or within the eye of the subject. In someembodiments, a medical device for measured or continuous release of SAPis embedded within tear duct or within the eyelid of a subject for thetreatment of DED, following or during, for example, reconstructivesurgery. The constant application of a low-viscosity solution of SAPenables deposition and spreading of the tear film, maintains the overallmakeup of the tear film to treat and prevent DED, and provide a visiblyclear artificial cover across the eye. Therefore, a medical deviceslowly releasing the SAP from the eyelid or tear duct serves as areservoir. A representation depicting the relative orientations of thetear duct and the cornea is provided in FIG. 1C.

In further embodiments, the methods include introducing to the surfaceof the eye, SAP containing a backing or support layers that providessupport and/or protection, such as a soft contact lens. The backing orsupport layer may be biodegradable or non-biodegradable and can becomposed of any material that is biocompatible for those embodiments,wherein the backing layer is also applied/implanted into the eye. Inother embodiments, the methods include the step of removing the backinglayer prior to application of the SAPs. The backing layer can beadhesive or non-adhesive. The backing layer can have associated with itone or more therapeutic, prophylactic, and/or diagnostic agents asdiscussed above. In some embodiments, the SAP are administered onto thesurface of the eye in the form of a contact lens, or as a coating orlayer associated with a contact lens or other implant that is placeddirectly onto the surface of the eye. Typically, the SAP contact thesurface of the eye upon administration of the lens. The contact lens canbe held in place for the desired amount of time, and replaced orrepeatedly applied as necessary, for example, until the desiredtreatment or prevention has been achieved. Therefore, in someembodiments, administration of SAP can occur via their controlledrelease from a contact lens. For example, the SAP would migrate into theinterface of the contact lens and the cornea where the SAP will thenstart to self-assemble or cause a non-adherent surface to be createdbetween the contact lens. This would allow for lens removal whileprotecting the corneal surface, for example, as the SAP wicks into theinterface between the contact lens and the cornea. Material wicked intothe interface subsequently may be able to penetrate the tissue anddeliver a bound cargo molecule to the interior of the eye or remain inthe interface between the lens and the tissue.

B. Diseases and Disorders to be Treated

SAP and compositions thereof, optionally including one or moreadditional therapeutic agents, can be used to treat or prevent any eyediseases or disorders according to the methods.

Symptoms of exemplary diseases, disorders, injuries and related symptomsthat affect the eye that can be treated include keratitis,conjunctivitis, DED and RCES, allergic conjunctivitis, exposurekeratopathy, amoebic keratitis, fungal keratitis, bacterial keratitis,viral keratitis, onchocercal keratitis, bacterial keratoconjunctivitis,viral keratoconjunctivitis, corneal dystrophic diseases, Fuchs'endothelial dystrophy, Stevens-Johnson syndrome, cornealneovascularization diseases, post-corneal transplant rejectionprophylaxis and treatment, autoimmune uveitis, infectious uveitis,anterior uveitis, posterior uveitis (including toxoplasmosis),pan-uveitis, an inflammatory disease of the vitreous or retina,endophthalmitis prophylaxis and treatment, macular edema, maculardegeneration, age related macular degeneration, corneal ectasia,keratoconus proliferative and non-proliferative diabetic retinopathy,hypertensive retinopathy, proliferative vitreoretinopathy, an autoimmunedisease of the retina, primary and metastatic intraocular melanoma,other intraocular metastatic tumors, open angle glaucoma, closed angleglaucoma, pigmentary glaucoma and combinations thereof. In someembodiments, the methods are effective to treat and prevent one or moresymptoms of an acute eye disease or disorder. In some embodiments, themethods are effective to treat and prevent one or more symptoms of achronic eye disease. In other embodiments, the methods are effective totreat and prevent one or more symptoms or disorders of the eye resultingfrom a traumatic or surgical injury. Therefore, the methods can be usedto treat and prevent conditions of the surface of the eye or one or moreof the interior compartments of the eye resulting from chemical burns,heat burns, surgery, injury, or other damage resulting to exposure toone or more environmental or chemical agents.

Treatment in Conjunction with Vitrectomy

For example, SAP may be used whether or not in conjunction withvitrectomy to address prophylactically or therapeutically to otherophthalmologic conditions, such as retinal detachment and proliferativevitreoretinopathy (PVR). Vitrectomy may be performed for a range of eyeapplications including to repair or remove a vitreous opacity, reductionof abnormal traction on the retina, for retinal surgery, for certainvitreoretinal diagnostic procedures, and to place a separate device ordrug.

Retinal detachment occurs when subretinal fluid accumulates in thepotential space between the neurosensory retina and the underlyingretinal pigment epithelium (RPE). Depending on the mechanism ofsubretinal fluid accumulation, retinal detachments traditionally havebeen classified into rhegmatogenous, tractional, and exudative. The mostcommon type is a rhegmatogenous retinal detachment, which occurs when atear in the retina leads to fluid accumulation with a separation of theneurosensory retina from the underlying retinal pigment epithelium.

SAP can be used to fill the vitreous cavity in lieu of or in combinationwith sterile saline (salt water) or with a vitreous substitute such as agas bubble or silicone oil for posterior eye procedures, such as tolessen traction on the retina, to close retinal tissue breaks andminimize bleeding in retinal repair, or prevent proliferativevitreoretinopathy (PVR) by providing a barrier to stabilize thestructure and minimize retinal pigment epithelial cell abnormalmigration and proliferation. Adequate density of SAP can be used tolessen constraints on head movement and position for the recoveringpost-operative surgical patient.

Other relevant conditions and procedures include, removal of vitreousopacities, separation of the vitreous from the retina, membrane peelingto lessen retinal traction, and placement of gas bubble. Barrierproperties may prevent neovascularization in diabetic retinopathy.

Prophylactic Treatment

The methods can be used for prophylactic treatment, for example, toprevent the onset or development of a disease or disorder of the eye. Insome embodiments, the methods include treating an individual who has notbeen diagnosed with one or more diseases or disorders of the eye, butwho has been identified as being at risk of developing or acquiring oneor more diseases, disorders or injuries of the eye. Exemplary subjectsat risk of developing or acquiring one or more diseases, disorders orinjuries of the eye include subjects with metabolic disease, at risk ofdeveloping infectious diseases, who are immunocompromised, orindividuals exposed to trauma, such as car wrecks, sports head injuries,and explosive devices. In some embodiments, the methods are used totreat or prevent disorders in diabetic subjects. In other embodiments,the methods are used to treat or prevent disorders in patients prior to,during or after one or more investigative or surgical procedures of theeye.

Ocular Inflammation

In some embodiments, SAP and compositions thereof are used to treat orprevent inflammatory responses within or around the eye. Therefore,methods of administering SAP to one or more of the structures of the eyefor treating and preventing ocular inflammation and/or intraocularinflammation are provided.

Suppression of unwanted inflammation resulting from disease and/orinjury can be of high clinical importance in many environments. Forexample, early and effective treatment of traumatic eye injuries may becritical to vision preservation. Self-assembling peptides and relatedpeptidomimetics (SAPs), a class of materials that can form abiocompatible barrier of intertwined nanofibers on contact with acharged surface such as a wound, appear to provide a solution for thesignificant unmet needs in corneal and other ocular structure healingafter acute eye injury in military settings.

Care protocols for ophthalmic injuries and diseases indicate that themost important medical actions for an ocular injury are evaluation ofthe extent of the injury, evacuation of the subject for more specializedtreatment, and immediate protection of the eye from further damage.

In some embodiments, SAPs immediately help to repair and/or mitigatefurther structural damage with a barrier that limits or preventsinflammation, infection and contamination, and creates amicroenvironment to enable restoration of the cornea, external andinternal globe structures, surrounding socket, optic nerve and relatedtissue.

Examination of an injured eye may be hampered by hemorrhage, tissuedamage or pain. SAP used shortly after injury during initial field-basedexamination can stop bleeding and allow better visualization of theinjury while relieving pain, protecting ocular structures andstabilizing the wound. In some embodiments, SAP is applied immediatelyfollowing trauma.

Dry Eye Disease (DED)

In some embodiments, SAP and compositions thereof are used to treat orprevent symptoms of DED, also known as keratoconjunctivitis sicca (Lemp,et al., Report of the International Dry Eye WorkShop (DEWS) Ocul Surf 5:65-204 (2007)), which is a multifactorial disease of the tears andocular surface that may cause discomfort, visual disturbance, tear filminstability and damage to the ocular surface. It is typicallyaccompanied by increased osmolarity of the tear film and inflammation atthe ocular surface. DED is associated with a range of symptoms and isoften further described according to its underlying cause, examples ofwhich include autoimmune, environmental, age-related, procedure-related(Lasik, etc.), and viewing related (reading, computer, movie,television, and driving) factors. In addition, adhesions may formbetween the palpebral conjunctiva of the eyelids and the cornealepithelium in DED patients, causing recurrent pain and further damage tothe corneal epithelium.

Treatment, which typically includes frequent applications per day ofartificial tears and non-prescription eye drops to mitigate irritationand lubricate the eyes, usually provides only minimal and limited reliefwithout modifying the disease course. DED is common, afflicting tens ofmillions of people globally.

In some embodiments, compositions of SAP are effective to treat andprevent one or more symptoms of DED by reducing the evaporation of tearsor tear film, preventing the formation of adhesions, providing acontinuous surface to maintain the tear film, enhancing or inducinghealing of damaged corneal tissue, reducing inflammation of the cornea,reducing irritation and pain, or combinations of these. In someembodiments, SAP are effective for the treatment and prevention of DEDassociated with Sjogren's syndrome.

Drug-Induced Eye Conditions

The formulations are also suitable for use in the management of eyeproblems that arise as a side effect of using one or more systemicdrugs. The SAP formulations are used prior, during or after taking oneor more systemic drugs. In some embodiments, SAP can be administeredinto or onto the eye prophylactically, to prevent disruption of the tearfilm or tear production in a subject identified at risk of disruption ofthe tear film or tear production. Therefore, methods of administeringSAP into or onto the eye for treating and preventing eye diseases,disorders, injuries and related symptoms that affect the eye in subjectswho have taken or are prescribed to take one or more therapeutic agentsassociated with disruption of the tear film or tear production areprovided. Exemplary drugs that can cause ocular side effects includecorticosteroids, antihistamines, antipsychotic medications,anti-malarial medications, blood pressure medications, herbal medicines,erectile dysfunction drugs, anticholinergics, immuno-suppressants,antibiotics, antiarrhythmic agents, and anti-cancer drugs/treatment.Some specific examples are bisphosphonate, amiodarone, tamsulosin,topiramate, ethambutol, minocycline, cyclosporine and tacrolimus.

Corticosteroids used for many conditions, such as asthma, allergies,arthritis and dermatitis, can cause swelling in the back of the eye orretina and may lead to cataracts. Antihistamines, used for conditionssuch as allergies, can raise certain patients' risk for glaucoma.Antipsychotic medications, such as THORAZINE® and MELLARIL® can be toxicto the retina. Anti-malarial medications, such as PLAQUENIL®(hydroxychloroquine), used to treat malaria, lupus and rheumatoidarthritis, is a known retinal toxin, and the effects are irreversible.FOSAMAX®, a bisphosphonate prescribed for post-menopausal women toprevent calcium bone loss, can cause orbital inflammation, uveitis andscleritis. Cyclosporine and Tacrolimus, commonly used in patients whohave undergone organ or bone marrow transplants, can cause posteriorreversible encephalopathy syndrome. These patients present withbilateral vision loss. Minocycline is a tetracycline derivative commonlyused to treat acne. Minocycline can cause increased intracranialpressure and papilledema, which can cause permanent vision loss if notreversed. Ethambutol is widely used to treat mycobacterial disease,including tuberculosis; if not taken at safe doses, it is an optic nervetoxin. Topiramate (Topamax) is used to treat epilepsy and migraineheadaches, and it is used off-label for weight loss. It can causeangle-closure glaucoma soon after starting treatment. Tamsulosin(Flomax), which is used to treat prostate enlargement and improveurinary flow in men. The well-known syndrome, intraoperative floppy irissyndrome, is often associated with men who were on medicine to relaxtheir prostate. Anticholinergics e.g., dicyclomine (BENTYL®), and otherdrugs with anticholinergic effects, are administered to patients whohave stomach conditions that require stomach relaxers and to patientswith Parkinson's disease. Young patients taking these drugs will developdifficulty with accommodation of the eyes. Erectile dysfunction drugs,e.g., sildenafil citrate (VIAGRA®) and tadalafil (CIALIS®)) are oftenprescribed for men with erectile dysfunction. Some ocular side effectsare blue vision, and ischemic optic neuropathy. Blood pressuremedications can cause glaucoma.

In some embodiments, the formulations and methods are used for treating,alleviating, and/or preventing one or more ocular symptoms that arise asa side effect from taking a systemic drug. In some embodiments, theformulations and methods are used for treating, alleviating, and/orpreventing one or more ocular symptoms in patients with ocular graftversus host disease. Ocular Graft Versus Host Disease (GVHD) occurs inpatients who have undergone allogenic hematological stem celltransplantation. It can occur in patients who have acute or chronicGVHD, though it is more common in patients with the chronic form.Approximately 40-90% of patients with chronic GVHD will develop ocularsymptoms.

Recurrent Corneal Erosion Syndrome (RCES)

SAP and compositions thereof are used to treat or prevent symptoms ofRDEC. RCES is a common clinical disorder characterized by a disturbanceat the level of the corneal epithelial basement membrane, resulting indefective adhesions and recurrent breakdowns of the epithelium. It mayarise spontaneously or from anterior basement membrane dystrophy (e.g.,Cogan dystrophy or map dot fingerprint dystrophy) or from otherproblems, such as adhesions between the palpebral conjunctiva of theeyelids and the corneal epithelium. Pain associated with RCE, whetherdue to trauma or to anterior basement membrane dystrophy, results fromabnormalities in the epithelial basement membrane.

Corneal erosions are common chronic ocular disorders and can worsen withtime, especially if neglected. RCES may occur either secondary tocorneal injury or spontaneously. When RCES arises spontaneously, apredisposing factor, such as corneal dystrophy, diabetes, or infection,may be the underlying cause. Management of RCES is usually aimed atregenerating or repairing the epithelial basement membrane to restorethe adhesion between the epithelium and the anterior stroma. However,current limited options for effectively controlling the development andprogression of RCES typically include application of lubricatingointment to prevent surface aggravation and antimicrobials to preventand reduce infection of the damaged mucosal tissue. Treatment can beprolonged and arduous, often leading to poor patient compliance poortherapeutic outcomes.

Although often unsuccessful, RCES treatment may include punctalocclusion, in which a plug is inserted into the tear duct, or directapplication of a bandage soft contact lens. Patients with refractoryRCES may undergo surgical interventions, such as Anterior StromalMicropuncture (ASM), phototherapeutic keratectomy and debridement of thecorneal epithelium, however, the attendant risks include scarring,glare, and blurred vision, and they often fail.

Epithelial basement membrane dystrophy is usually bilateral andcharacterized by various patterns of dots, parallel lines that mimicfingerprints, and patterns that resemble maps, which appear in theepithelium. Individual microcysts may be oval, oblong, or comma-shapedand rarely appear alone but usually are associated with map andfingerprint patterns. On the other hand, the map and fingerprintpatterns appear without dots or individual microcysts. Map andfingerprint alterations of the corneal epithelium are not rare and canbe found in asymptomatic individuals without prior history of trauma orocular disease. Epithelial changes are more common than previouslyrecognized and are frequently present in conditions involving cornealedema (e.g., such as near a healing cataract surgery incision) or in thecenter of the cornea associated with Fuchs corneal dystrophy.

The epithelial healing process begins when basal epithelial cellsundergo mitosis, producing new cells that occupy fresh wounds. Basalcells adhere the epithelium to the stroma in two ways: they secrete thebasement membrane, and they contain hemidesmosomes, which behave aslinchpins that protrude through the posterior surface of basal cells andinto the stroma; each is held in place by an anchoring fibril. Anydisruption to basal cell production makes the eye more prone torecurrent erosion.

RCES occurs because of a defect in the epithelial basement membrane andin hemidesmosomes formation, resulting in epithelial loss, microcysts,and bullae. RCES occurring after corneal injury or insult typicallyresults from improper or inadequate healing of the basement membrane,typically either because the basal epithelial cells fail to produceproper basement membrane complexes to attach to the Bowman layer andstroma or because of faulty basement membrane adherence.

Prognosis for RCES due to trauma is better than RCES that arises thespontaneously. The disease process underlying spontaneous RCES may beepithelial basement membrane corneal dystrophy. Electron microscopestudies of tissue during RCES episodes show separation of the anchoringsystem at the level of the epithelial cell membrane or below the levelof the anchoring plaques. Normal and degenerate polymorphonuclearleucocytes (PMNs) are found within and between the epithelial cells andwithin the anchoring layer. The degenerate PMNs may secretemetalloproteinases that cleave the Bowman layer below the anchoringsystem.

In some embodiments, compositions of SAP are effective to treat andprevent one or more symptoms of RCES by reducing the evaporation oftears or tear film, preventing the formation of adhesions, providing acontinuous surface to maintain the tear film, enhancing or inducinghealing of the damaged corneal tissue, reducing inflammation of thecornea, reducing irritation and pain, or combinations of these. In aparticular embodiment, compositions of SAP are effective to treat andprevent one or more symptoms of RCES by enhancing or inducing theregeneration or repair of the epithelial basement membrane to restorethe adhesion between the epithelium and the anterior stroma. Thisenhancement can be achieved by forming a barrier to inflammatory cellsand markers, and limiting access of inflammatory factors, such asmicrobial or environmental contaminants, to the damaged corneal surface.

Fuchs' Dystrophy

In some embodiments, formulations of SAP are used for treatment orprevention of one or more of the symptoms associated with Fuchs'Dystrophy.

Fuchs' Dystrophy (also known as Fuchs' corneal endothelial dystrophy orFCED) is a slowly progressing corneal dystrophy that usually affectsboth eyes. FCED results in corneal clouding, loss of corneal sensationand the formation of epithelial bullae. Multiple stages of Fuchs'endothelial dystrophy are recognized, typically evolving gradually overa period of about 25 years.

The first stage is the onset of cornea guttata, usually in the fourthdecade of life. Subjective symptoms rarely occur until the fifth orsixth decade. During the asymptomatic phase, endothelial guttata andpigment dusting can be seen by slit lamp examination of the centralcorneal endothelium and by specular reflection. The guttata excrescencescan become more numerous and confluent so that individual guttata arelost completely in the beaten-metal appearance of the endothelialsurface. The central cornea is involved first, and, as the diseaseprogresses, it spreads toward the periphery.

In the second phase of the disease, blurred vision, glare, and halosaround lights develop because of incipient corneal edema in the stromaand epithelium. Epithelial edema can be seen as small droplets(bedewing) on retroillumination with the slit lamp. Epithelialmicrocysts coalesce to form bullae, which produce varying amounts ofpain when they burst; hence, the name bullous keratopathy. Striae formin the Descemet membrane as the cornea thickens posteriorly due tostromal swelling. The arc of the Descemet membrane from limbus to limbusis shortened, causing wrinkles in the Descemet membrane called striae.The microcystic epithelial vesicles may break, causing foreign bodysensations and severe pain with more extensive corneal epithelialdisruption.

In the third stage, RCES, microbial ulceration, and persistent pain mayoccur. Corneal sensitivity usually is reduced.

Eye Infections

The formulations are suitable for use in the management of eyeinfections. Therefore, in some embodiments, SAP and compositions thereofare used to treat or prevent eye infections from bacteria, fungi,viruses, and protozoa. Eye infections can occur in different parts ofone or both eyes and can be associated with conjunctivitis, stye,inflammation pain, etc.

In some embodiments, the formulations are for prophylactic purposes toprevent onset of an infection. For example, people recently exposed to aperson with an eye infection, e.g., conjunctivitis, can use SAP forprophylactic purposes. In some embodiments, the formulations are used toreduce or alleviate one or more symptoms from an eye infection.

In some embodiments, SAP and compositions thereof are used to treat orprevent eye keratitis, i.e., inflammations of the cornea, by applyingtopically or by injection the compositions containing one or more SAPinto the corneal stroma. Conditions treated by the methods include, butare not limited to, amoebic keratitis, fungal keratitis, bacterialkeratitis, viral keratitis, and onchorcercal keratitis.

In some embodiments, SAP and compositions thereof are used to treat orprevent amoebic keratitis. Amoebic keratitis can be caused byacanthamoeba and can be treated by administering to the corneal stromaof a subject in need of treatment thereof the compositions, optionallycontaining one or more anti-amoebic agents. Any anti-amoebic agent knownin the art can be combined with the compositions. Representative,anti-amoebic agents include, but are not limited to, polyhexamethylenebiguanide (PHMB), propamidine isethionate, miconazole nitrate, neomycin,chlorhexidine digluconate, polymyxin B, clotrimazole, and combinationsthereof.

In some embodiments, SAP and compositions thereof are used to treat orprevent fungal keratitis. Fungal keratitis can be caused by, forexample, aspergillus fumigates, fusarium, and/or yeasts, such ascandida, and can be treated by administering to the corneal stroma of asubject in need of treatment thereof the compositions, optionallycontaining one or more antifungal agents. Any antifungal agent known inthe art can be combined with the compositions. Representative,antifungal agents include natamycin, nystatin, amphotericin B,chlorhexidine, fluorinated pyrimidines, such as flucytosine, azoles,such as imidazoles and triazoles, including ketoconazole, miconazole,itraconazole, fluconazole, econazole, and clotrimazole, and combinationsthereof.

In certain embodiments, SAP and compositions thereof are used to treator prevent bacterial keratitis. Bacterial keratitis can be caused, forexample, by streptococcus, pseudomonas, Enterobacteriaceae (includingKlebsiella, Enterobacter, Serratia, and Proteus), and staphylococcusspecies. Such infection can be treated by administering to the cornealstroma of a subject in need of treatment thereof the compositions,optionally containing one or more anti-bacterial agents. Further, up to20% of cases of fungal keratitis (particularly candidiasis) can becomplicated by bacterial co-infection. Thus, in some embodiments, themethods include combination therapies involving administering to thecorneal stroma of a subject in need of treatment thereof thecompositions, optionally containing one or more antifungal agents andone or more anti-bacterial agents.

In certain embodiments, SAP and compositions thereof are used to treator prevent viral keratitis. Viral keratitis can, for example, be causedby a herpes simplex virus and can be treated by administering to thecorneal stroma of a subject in need of treatment thereof thecompositions, optionally containing one or more anti-viral agents, suchas, but not limited to, acyclovir. Any anti-viral agent known in the artcan be combined with the compositions.

In some embodiments, SAP and compositions thereof are used to treat orprevent keratitis associated with infection by a nematode, such asonchoceral keratitis. Onchoceral keratitis, also referred to as “RiverBlindness,” is caused by the nematode Onchocerca volvulus and can betreated by administering to the corneal stroma of a subject in need oftreatment thereof the compositions, optionally containing one or moreanti-parasitic agents, such as, but not limited to ivermectin. Anyanti-parasitic agent known in the art can be combined with thecompositions.

C. Effective Amounts and Controls

SAP can be administered to the eye therapeutically to achieve atherapeutic benefit, or prophylactically to achieve a prophylacticbenefit, or to achieve both a therapeutic and prophylactic benefit.Therapeutic benefit means treating the underlying disorder includingeradication or amelioration of one or more of the symptoms associatedwith the underlying disorder such that the patient reports animprovement in feeling or condition, notwithstanding that the patientcan still be afflicted with the underlying disorder. For example,administration of a composition to a patient suffering from a conditionprovides therapeutic benefit when the patient reports a decrease in theseverity or duration of the symptoms associated with the condition.Therapeutic benefit also includes halting or slowing the progression ofthe disease, regardless of whether improvement is realized by thepatient.

In preferred embodiments, SAP are retained at the site of administration(e.g., at the surface of the cornea) for periods of time sufficient toyield a therapeutic effect. For example, when administered topicallyonto the cornea, compositions of SAP assemble at the surface of thecornea to form an SAP structure that resists clearance by tearing, eyemovement and diffusion at the site of the administration. In preferredembodiments, the amount of time that the SAP remains in contact with thesurface of the cornea is sufficient to prevent or reduce cornealinflammation and edema. In certain embodiments, the ability to resistclearance from the site of administration and prevent or reduce cornealinflammation is associated with parameters such as the concentration andhomogeneity of SAP within the composition.

Typically, SAP are administered to the eye in an effective amount inorder to achieve a clinically significant result. Effective dosages andconcentrations are those that provide a therapeutic benefit and can bedetermined according to the desired therapeutic or prophylactic result.For example, the SAP can be effective to treat and prevent one or moresymptoms of an ocular disease or disorder. Exemplary symptoms that canbe treated, prevented reduced or otherwise moderated by SAP include, butare not limited to, pain, irritation, inflammation and swelling of theeye, redness or discoloration of the eye and/or surrounding tissues,dryness of the eye, discharge and/or watering of the eyes, impairedvision, blindness, and photosensitivity.

In one embodiment, the SAP are administered in an amount effective toreduce or prevent inflammation of the eye. In some embodiments, the SAPare administered in an amount effective to reduce or preventinflammation of the cornea or surface of the eye. Reducing inflammationcan include reducing or preventing tissue damage, inducing or enhancinghealing, reducing or preventing scarring, or combinations of these.

In some embodiments, the SAP are in an amount effective to provide afluid impermeable barrier at the site of administration. The barrier iseffective to prevent the movement of bodily fluids and contaminantsthrough the structure. When SAP provide a barrier at the surface of theeye, the presence of the barrier can be effective to treat diseasesassociated with recurrent infection or contamination of a damaged ormissing corneal tissue. For example, in some embodiments, the presenceof a self-assembled barrier provides a continuous surface that preventsthe formation of adhesions between the damaged corneal epithelium andother tissues. In other embodiments, the presence of the barriermitigates inflammatory processes associated with eye diseases, such aschronic eye diseases (e.g., RCES).

In some embodiments, the SAP are in an amount effective to provide afluid permeable structure at the site of administration. In someembodiments, the presence of a fluid permeable structure that acts as amembrane to enable passage of one or more fluids into and through thestructure provides a suitable scaffold for the replacement, repair oraugmentation of a diseased or damaged tissue on the surface of the eyeor within the eye.

For guidance, one can consult texts such as Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10th Ed., and Katzung, Basic andClinical Pharmacology.

Inflammation and intraocular pressure (TOP) can be measured before andafter administration of the compositions by methods known in the art.For example, a Reichert Tono-Pen contact tonometer can be used to assessthe IOP, and changes in IOP. A subject, for example, a human patient,can be evaluated on several post-administration days, for example at 1,7, 14 and 30 days post-administration, using the same evaluationprocedure on each day. One or more measurements, for example about five,can be obtained for each subject at each time point. When measuring IOPaccording to methods known in the art, for example the method above, IOPis shown to be reduced by, for example, at least about 10%, or at leastabout 30%, or at least about 50% over what is observed when a controlcomposition, for example a composition that does not include SAP, isadministered to the patient. To measure inflammation, slit-lampbio-microscopy can be performed to examine structures of the eye, forexample, the cornea, for signs of inflammation.

The examination can involve observation of criteria such as the presenceof cells, flare and fibrin. The subjects can be evaluated on severalpost-injection days, for example at 1, 7, 14 and 30 days post-injectionusing the same evaluation procedure on each day. After general and/orlocal anesthesia is achieved, each subject can be examined for grossabnormalities. The exams can be performed by the same trainedophthalmologist, and the ophthalmologist can be blinded to theassignment of the treatment and control subjects.

Quantification of inflammation of an eye compartment such as the corneacan be performed using a modified version of the Standard UveitisNomenclature clinical grading scheme. When measuring inflammationaccording to methods known in the art, for example the method above,inflammation is shown to be reduced by, for example, at least about 10%,or at least about 30%, or at least about 50% over what is observed whenan equivalent control composition, e.g., lacking SAPs, is administeredto the patient. When comparing an TOP or inflammation measurementobtained after administration of SAP versus an TOP or inflammationmeasurement obtained for a control composition, the measurementscompared can, and frequently should, be taken at similar time pointspost administration. For example, an TOP measurement afteradministration of SAP taken 7 days post-administration is generallycompared with an TOP measurement for a control composition taken 7 dayspost-administration.

All references cited herein are incorporated by reference in theirentirety. The present description will be further understood byreference to the following non-limiting examples.

Example 1: SAP Exhibit Therapeutic Effects in the Eye

The anti-inflammatory effect of SAP was examined using thelipopolysaccharide ocular inflammation model, and measurement ofmicroglial cell activation was assessed as an indicator of inflammation.Morphological changes in macrophage cells, such as microglial cells, canbe used as a marker of the presence and extent of inflammation. Methodsfor identifying and scoring variations in microglial cell morphology inresponse to inflammatory stimuli are described in the art (see, forexample, Jonas, et al., PLoS ONE 7(2): e30763 (2012)).

Methods and Materials

Animals

Adult white Sprague-Dawley rats weighing between 200 and 240 grams werehoused in a 12 hour light/12 hour night cycle with free access to foodand water. The animals were randomly divided into study or controlgroups.

Intravitreal Injections

Animals were anesthetized by an intraperitoneal injection of xylazine 2%(5 mg/kg body weight) and ketamine 10% (100 mg/kg body weight). Topicalanesthesia was additionally provided by repeated administration ofproparacaine (0.5%) drops onto the cornea. A thorough disinfection ofthe ocular surface, the lid margins and the periocular skin was carriedout with iodine 5% solution.

Using a glass pipette pulled to the size of a 30 gauge needle andattached to a syringe, 2 μl of sterile, pyrogen-free saline containinglipopolysaccharides from Salmonella typhimurium (catalog no. L-7261;Sigma-Aldrich, St. Louis, Mo.) was intravitreally injected.

In a preliminary experiment, the optimal lipopolysaccharide (LPS)concentration and survival time was assessed in a group of animals.Concentrations of 1 μg or 2 μg LPS/μL were administered, and the animalssacrificed after a post injection survival time of 8, 12, 24, 72 hours,1 week, or 2 weeks, respectively. This study showed that the optimalconcentration was 2 μg LPS/μL in 2 μl of carrier solution, and that asurvival time of 66 to 68 hours was the optimal time for measurement andquantification of retinal inflammation. Retinal inflammation and retinaldestruction with tissue lysis was also detected starting at about 24hours post injection. The retinal lysis led to an accumulation ofretinal ganglion cells in the pre-retinal vitreous.

For the primary experiment, the study included 40 Sprague-Dawley rats.All intravitreal injection volumes were of 2 μl, to reduce the effectsof increased ocular pressure. All intravitreal injections oflipopolysaccharides (LPS) volumes were 4 μg at a concentration of 2μg/μL.

The control groups (n=20) received either an intravitreal salineinjection (n=5), lipopolysaccharide (LPS) alone (n=15), or no injectionas a normal control group (n=5). Animals in the SAP study group (n=15)also received the self-assembling peptide (amino acid sequenceRADARADARADARADA; “RADA-16”) in concentrations of 0.5% of SAP/2 μL.

At one, three and seven days after the injections, the animals weresacrificed, and the globes were immunohistochemically stained andhistomorphometrically examined. The contralateral eye from the LPS groupwas also examined.

Immuno-Histochemical Staining

The eyes were embedded with Optimal Cutting Temperature compound(Tissue-Tek® O.C.T., Ted Pella, Inc. Redding, Calif.) after the lenseswere removed. The globes were sectioned and placed on subbed slides.After being washed in 0.01M phosphate buffered saline for 10 minutes,the sections (15 μm) were immersed in a blocking solution containing0.3% triton, 2.5% bovine serum albumin (BSA; Proliant Co, Ankeny, Iowa,USA) and 2% goat serum for 30 minutes. The sections were incubated in aprimary antibody diluted solution overnight at 4° C., containing rabbitanti-IBA1 (Ionized calcium Binding Adaptor molecule 1; 1:500; Wako PureChemical Industries, Ltd. Osaka, Japan) and mouse anti rat CD 68 (ED1)(1:1000; AbD Serotech, D-40470 Dusseldorf, Germany). The sections werethen washed in 0.01M phosphate buffered saline (10 minutes, 3 times),and incubated with secondary antibody diluted solution at roomtemperature for 2 hours (Goat-anti-rabbit 568, Goat-anti-mouse Alexa488, (Invitrogen, Carlsbad, Calif., 1:400)). The slides were then washedin 0.01M phosphate buffered saline (10 minutes, 3 times) and coverslipped with fluorescein mounting medium with DAPI(4′,6-diamidin-2′-phenylindol-dihydrochloride; Dako Ltd, Glostrup,Denmark). All images were taken under a 20× objective with a Carl ZeissFlour microscope (Carl Zeiss Inc. Oberkochen, Germany) fitted with aspot camera. All illumination levels were fixed during imageacquisition.

To determine a potential interaction between the LPS and RADA-16, a SDSPAGE gel electrophoresis (sodium dodecylsulfate polyacrylamide gelelectrophoresis) was performed in a 12.5% gel, running 4.5 h with ureain stacking and separating, 5% SDS and 5% urea in loading buffer.

Assessment of Intraocular Inflammation

The amount of intraocular inflammation was assessed by quantification ofimmunohistochemically labeled activated retinal microglial cells, whichfunction as the immune mediators of the central nervous system.

The images of the retinas were quantified using the program Image J fromthe National Institute of Health. Continuous sections of the retina,choroid and pre-retinal vitreous were outlined, the number of activatedwas determined, and the density of activated pixels was calculated.These measurements were performed in a masked manner.

Six categories of inflammation have been described in the microgliasystem. Each retinal section was examined, and each microglia wascategorized based on activation level (type 1-6) according tomorphological characteristics. In addition, both the microglia markerionized calcium-binding adapter molecule 1 (IBA-1) and the macrophagemarker ED-1 immuno-histochemical reactivities were used as indicators ofthe shift from type 4 to 5 activation level. Three blind assessors wereused to count and categorize the microglial activation level. A weightedcalculation based on the morphological classification of activatedmicroglia cells (the “JEB” score, as determined according to the methodsof Jonas, et al., PLoS ONE 7(2): e30763 (2012)) was derived and appliedto the activation category level to provide an inflammation score thatcould indicate anti- or pro-inflammatory effects, and the results wereaveraged across the three assessors (see Table 4). Normalization wasdone by dividing the number of microglia over the volume of thesections. A potential direct interaction between the lipopolysaccharides(LPS) and the SAP material was examined by gel electrophoresis.

Results

The density of activated retinal microglial cells was significantlylower in animals in the SAP group compared to LPS controls. The numbersof activated microglia within the tissue sections from each groupassessed are indicated in Table 4 below.

TABLE 4 Results across animals treated with LPS, LPS + AC5 (RADA),Saline, and untreated controls. TOTAL AREA TOTALS FOR COMPARISON JEB #WEIGHT N SD SD NORMAL −0.513244048 −3.190105616 5 1.227546 9.255464SALINE DAY 3 5.309575517 30.81892728 5 4.217035 25.37896 CONTRALATERALEYE LPS DAY 3 3.583019943 15.15861947 10 3.927901 23.80311 1 DAY LPS99.33963341 787.1193889 5 26.36615 236.5959 3 DAY LPS 145.0817385818.3299501 10 31.73665 116.6731 0.5% RADA + LPS DAY 1 11.6326334874.99464859 5 13.08403 26.60439 0.5% RADA + LPS DAY 3 8.50214947155.91265281 5 6.457764 45.92137 0.5% RADA + LPS DAY 7 9.15848169252.65745506 5 8.855492 47.74718

The amount of inflammation in the experimental combination injectionswith both SAP and LPS showed no significant difference from normal orsaline injected controls (0.5% SAP+LPS [Day 1, 75.0±26.6; Day 3,55.9±45.9; Day 7, 52.7±47.7], saline injection [Day 3, 30.8±25.4],normal control [−3.2±9.3]). In the LPS-only group, there was asignificant increase in inflammation (Day 1, 787.1±236.6; Day 3,818.3±116.7). The level of inflammation was significantly (P<0.001)lower in the SAP group than in the LPS-only control group. Thecontralateral eye of the LPS-only group showed evidence of a numerical,but not significant increase in the inflammation score (Day 3,15.16±23), compared to the normal non-injected eye. Electrophoresisrevealed no interaction between the lipopolysaccharides and SAP.

Differences in the density (mean±standard deviation) of activatedretinal microglial cells in the LPS control and RADA study group aredepicted as histograms in FIGS. 2 and 3.

In this experimental intraocular inflammation model of injected LPS, theadditional intraocular application of SAP along with LPS was associatedwith a marked reduction in signs of retinal inflammation. The density ofactivated retinal microglial cells was significantly lower in the eyesof the study animals with the combined application oflipopolysaccharides and SAP than in the eyes of the LPS-only controlgroup.

Example 2: (RADA)₂ and EARA Based SAPs Exhibit Therapeutic Effects inthe Eye Methods and Materials

Animals

Adult white Sprague-Dawley rats with a weight of between 200 and 240grams were housed in a 12 hours light/12 hours night cycle with freeaccess to food and water. The animals were randomly divided into a studyor control groups.

Treatment and Analysis

Intravitreal injections, immunohistochemical staining and assessment ofintraocular inflammation were generally performed as described inExample 1.

1 μl sterile lipopolysaccharide (LPS; from S. typhimurium; catalog no.L-7261; Sigma-Aldrich, St. Louis, Mo.) either alone or in combinationwith (RADA)₂ (SEQ ID NO: 88) solutions at 0.1%, 1.0%, and 10.0% wereadministered via direct intravitreal injections. The animals weresacrificed 24 hours after treatment. The experiments were repeated atleast 5 times.

In some experiments, different SAP, including S2 (H₂N(EARA)COOH; SEQ IDNO: 92), S3 (H₂N(RARA)CONH₂; SEQ ID NO: 413), and S5 (H₂N(EARA)₂CONH₂;SEQ ID NO: 414), were evaluated as described above for (RADA)₂ (SEQ IDNO: 88).

Finally, a conjugate of an EARA based SAP and a tissue-specific motif(MSCRAMM; SEQ ID NO: 141) was also evaluated using thislipopolysaccharide ocular inflammation model. Experiments were performedas described above for (RADA)₂ (SEQ ID NO: 88). Briefly, 1 μl sterilelipopolysaccharide (LPS; from S. typhimurium; catalog no. L-7261;Sigma-Aldrich, St. Louis, Mo.) either alone or in combination withMSCRAMM(EARA)₄ (SEQ ID NO: 432) solutions at 1.0%, and 3.0% wereadministered via direct intravitreal injections. The animals weresacrificed 24 hours after treatment. The experiments were repeated threetimes for each SAP conjugate concentration (that is, n=3 for 1%; n=3 for3%).

Results

As shown in FIG. 4, at all concentrations of (RADA)₂ (SEQ ID NO: 88),the combinatorial application of (RADA)₂ along with LPS was associatedwith a marked reduction in signs of retinal inflammation compared tocontrol. The density of activated retinal microglial cells wassignificantly lower in the eyes of the experimental group (combinationinjection with both (RADA)₂ and LPS) compared to the controls(injections of LPS alone).

Furthermore, the combinatorial application of EARA (SEQ ID NO: 92),H₂N(RARA)CONH₂ (SEQ ID NO: 413), or H₂N(EARA)₂CONH₂ (SEQ ID NO: 414)along with LPS was associated with a marked reduction in signs ofretinal inflammation compared to control (FIG. 5). In particular, S3(H₂N(RARA)CONH₂; SEQ ID NO: 413) reduced LPS induced inflammation tonormal levels (FIG. 5).

Application of the MSCRAMM(EARA)₄ (SEQ ID NO: 432) conjugate was alsoassociated with a reduction in signs of retinal inflammation compared tocontrol (LPS only). There appeared to be no inflammation and no evidenceof ED1 reactivity in MSCRAMM(EARA)₄ treated animals. Any activation ofmicroglia for MSCRAMM(EARA)₄ treated animals was a 4 or below (JEBscore), while animals treated with LPS alone exhibited microglialactivation of 5 and above.

The data demonstrate that compositions of SAP, and conjugates thereof(e.g., (RADA)₂, SEQ ID NO: 88; EARA, SEQ ID NO: 92; H₂N(RARA)CONH₂, SEQID NO: 413; H₂N(EARA)₂CONH₂, SEQ ID NO: 414; MSCRAMM(EARA)₄, SEQ ID NO:432) can be used to treat eye diseases (e.g., reducing associatedsymptoms, such as retinal inflammation).

Example 3: Delivery of SAP to the Retina Via Application of Eye Drops

The fate of SAP upon application to the eye was investigated.Specifically, the ability of SAP to reach the retina via application ofeye drop solutions of the SAP to the cornea was evaluated using anSAP-Cy5 conjugate.

Methods

Animals

Adult white Sprague-Dawley rats weighing between 200 and 240 grams werehoused in a 12 hour light/12 hour night cycle with free access to foodand water. The animals were randomly divided into experimental orcontrol groups.

Conjugation of Cy5.5 to RADA

Conjugates of Cy5.5 and RADA (SEQ ID NO: 57), Cy5.5 and RADARADA((RADA)₂; SEQ ID NO: 88), and Cy5.5 and H₂N(RADA)₄CONH₂ (SEQ ID NO: 433)were prepared according to a modified protocol of Chen X., et al.,Cancer Res., 64(21): 8009-8014 (2004).

In one experiment, RADA-4-Cy5.5 conjugate was prepared as follows: 0.5mg of H₂N(RADA)₄CONH₂ was dissolved in 50 μL of DMSO to a finalconcentration of 0.5% in DMSO. The solution was mixed with 20 μL ofdiethyl isopropyl amine (DIPEA) to adjust the pH to 12. A solution ofCy5.5 dyes (1.0 mg in 20 μL of DMSO) was added at room temperature forconjugation and the mixture was periodically vortexed over 2 hours.

TABLE 5 Components for Cy5.5(RADA)₄ conjugation Weight Final VolumeFinal Conc Final Conc Mole Sample Formula (mg) (mL) (μg/μL) (mM) ratioSEQ ID NO: 433 H₂N(RADA)₄CONH₂ 0.5 0.1 5 2.994 1 Cy5.5 1.0 0.1 10 8.4252.8

In some experiments, the RADA-4 solution was prepared with water as thesolvent, for example: to a solution of RADA-4 (1.8 mg of H₂N(RADA)₄CONH₂(SEQ ID NO: 433) in 500 μL of Milli-Q water), 1 μL of Diisopropylethylene amine (DIPEA) was added to obtain pH 8 from 4. To prepare aworking Cy5.5 stock, 165 μL of Milli-Q water was added to 165 μL Cy5.5to make a 6.59×10⁻⁷ M solution. The final conjugate was prepared bymixing equal volumes (165 μL) of the buffered (pH 8) (RADA)₄ solutionand Cy5.5 solution at room temperature for 2 hours.

TABLE 6 Components for Cy5.5(RADA)₄ conjugation Weight Milli-Q Vol.Final Conc. Mole Mole Sample Formula (mg) (μL) (μg/μL) (×10⁻⁷) ratio SEQID NO: 433 H₂N(RADA)₄CONH₂ 1.5 500 3 8.98 1.3 Cy5.5 1.0 500 2 13.17 1.0

The Cy5.5 SAP conjugates listed in Table 7 were also prepared andevaluated as described above for the RADA-based conjugates.

TABLE 7 Cy5.5 SAP conjugates Cy5.5(RADA)₄CONH₂ SEQ ID NO: 434Cy5.5(EARA)CONH₂ SEQ ID NO: 435 Cy5.5(EARA)COOH SEQ ID NO: 436Cy5.5(RARA)CONH₂ SEQ ID NO: 437 Cy5.5(RARA)COOH SEQ ID NO: 438Cy5.5(EARA)₂CONH₂ SEQ ID NO: 439 Cy5.5(EARA)₂COOH SEQ ID NO: 440Cy5.5(RARA)₂CONH₂ SEQ ID NO: 441 Cy5.5(RARA)₂COOH SEQ ID NO: 140Cy5.5(RADA)COOH SEQ ID NO: 442 Cy5.5(RADA)CONH₂ SEQ ID NO: 443Cy5.5(RADA)₂COOH SEQ ID NO: 444 Cy5.5(RADA)₂CONH₂ SEQ ID NO: 445

Treatment

Eye drops were applied to both eyes of rats. The experimental group ofrats were administered 2 μL of Cy5.5-(RADA)₄ conjugate via drops, whilecontrol rats were administered Cy5.5 only via drops. The experimentswere repeated using Cy5.5 conjugated to RADA (Cy5.5(RADA)COOH; SEQ IDNO: 442) and Cy5.5 conjugated to (RADA)₂ (Cy5.5(RADA)₂COOH; SEQ ID NO:444).

The rats were sacrificed 24 hours or 3 days post treatment. The eyeballs of the rats were harvested and were then fixed in 4%paraformaldehyde (PFA) overnight. Excessive PFA was washed with 0.1Mphosphate buffer (0.1MPB) 3 times (30 minutes each). The eyeballs werethen dehydrated with 30% sucrose solution (30 g sucrose in 100 mL of 0.1MPB) overnight, while replenishing the sucrose solution once for atleast 6 hours. As necessary, the shape of the eyeballs was reformed withO.C.T. (Optimal Cutting Temperature compound; TISSUE-TEK® O.C.T.; CA) byinjecting through the optic nerve. Each eye was embedded entirely in amount with O.C.T. in liquid nitrogen to form frozen blocks. Blockscontaining the eye samples were then sectioned at 20 microns and wereplaced on charged slides and allowed to dry. The slides were then rinsedto remove excess OCT and cover-slipped. The fate of the Cy5.5-RADAconjugate was investigated by tracing the color of the Cy5.5 in the eye.Visualization was performed with light microscopy and photographs weretaken with a SPOT camera affixed to the Zeiss microscope.

Results

At both 24 hour and 3 day time points, pink coloration was observedthroughout the outer retina of mice treated with the Cy5.5-(RADA)₄conjugate. However, no pink coloration was observed in any part of theretina or optic nerve head of control rats (i.e., receiving Cy5.5 only)at either time point. This data demonstrates that upon application aseye drops, the SAP can penetrate the cornea and migrate to the retina.

SAP can be used for treatment of inflammation from diseases in theretina or other parts of the eye, such as but not limited to, diabeticretinopathy, retinitis pigmentosa, uveitis, inflammatory eye diseases,autoimmune eye diseases, Reiter's syndrome, psoriasis, rheumatoidarthritis, Sjogren's Syndrome, optic neuritis, and sarcoidosis. In suchapplications, the compositions could be applied as drops. It iscontemplated that the compositions containing SAP could be used fortreatment and/or repair of damaged tissue, e.g., retina.

We claim:
 1. A dosage unit of a formulation suitable forophthalmological administration comprising self-assembling peptides orself-assembling peptidomimetics in an amount effective for the treatmentor prevention of one or more diseases, injuries, or symptoms of diseasesor disorders of the eye.
 2. The dosage unit of claim 1, wherein the oneor more self-assembling peptides or self-assembling peptidomimetics havea sequence of amino acid residues conforming to one or more of FormulasI-XII:((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n)  (I)((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n)  (II)((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n)  (III) and((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n)  (IV)Xaa^(neu)((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n)  (V)Xaa^(neu)((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n)  (VI)((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n)Xaa^(neu)  (VII)((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n)Xaa^(neu)  (VIII)((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n)Xaa^(neu)  (IX)((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n)Xaa^(neu)  (X)Xaa^(neu)((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n)  (XI)Xaa^(neu)((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n)  (XII) whereinXaa^(neu) represents an amino acid residue having a neutral charge; Xaa⁺represents an amino acid residue having a positive charge; Xaa⁻represents an amino acid residue having a negative charge; x and y areintegers having a value of 1, 2, 3, or 4, independently; and n is aninteger having a value of 1-5.
 3. The dosage unit of claim 1, whereinbetween about 70% and 100% of the self-assembling peptides orself-assembling peptidomimetics are of the same size and have the sameamino acid sequence.
 4. The dosage unit of claim 1, further comprising apH-adjusting agent.
 5. The dosage unit of claim 1, wherein theconcentration of self-assembling peptides or self-assemblingpeptidomimetics in the formulation is between about 0.1% w/v and about6% w/v, inclusive, preferably between about 0.1% w/v and about 4% w/v,inclusive.
 6. The dosage unit of claim 1, wherein the concentration ofions in the formulation is between 5 nM and less than 5 mM, preferablyless than 10 mM, and most preferably less than 5 mM.
 7. The dosage unitof claim 1, comprising a pharmaceutically acceptable excipient foradministration into or onto the eye.
 8. The dosage unit of claim 1,wherein the composition is a device comprising a backing material orsupport structure.
 9. The dosage unit of claim 1, further comprising onemore therapeutic agents, prophylactic agents, diagnostic agents, orcombinations thereof.
 10. The dosage unit of claim 1, wherein the dosageunit is in a form selected from the group consisting of powders,liquids, emulsions, gels, wafers, tablets, nanoparticles, andmicroparticles.
 11. The dosage unit of claim 1, wherein the dosage unitis, a device selected from the group consisting of ocular implants,intraocular lens, contact lens, coatings on a medical device, eyepatches, and woven or non-woven fiber wound coverings.
 12. The dosageunit of claim 1, wherein the composition is dried, dehydrated or vacuumpackaged.
 13. The dosage unit of claim 1, wherein the composition isformulated for administration into the eyes in the form of eye drops.14. The dosage unit of claim 1 in a kit with means for administration.15. A contact lens or intraocular implant comprising self-assemblingpeptides or self-assembling peptidomimetics having a sequence of aminoacid residues conforming to one or more of Formulas I-XII:((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n)  (I)((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n)  (II)((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n)  (III) and((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n)  (IV)Xaa^(neu)((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n)  (V)Xaa^(neu)((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n)  (VI)((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n)Xaa^(neu)  (VII)((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n)Xaa^(neu)  (VIII)((Xaa^(neu)-Xaa⁺)_(x)(Xaa^(neu)-Xaa⁻)_(y))_(n)Xaa^(neu)  (IX)((Xaa^(neu)-Xaa⁻)_(x)(Xaa^(neu)-Xaa⁺)_(y))_(n)Xaa^(neu)  (X)Xaa^(neu)((Xaa⁺-Xaa^(neu))_(x)(Xaa⁻-Xaa^(neu))_(y))_(n)  (XI)Xaa^(neu)((Xaa⁻-Xaa^(neu))_(x)(Xaa⁺-Xaa^(neu))_(y))_(n)  (XII) whereineach Xaa^(neu) represents an amino acid residue having a neutral charge;Xaa+ represents an amino acid residue having a positive charge; Xaa−represents an amino acid residue having a negative charge; x and y areintegers having a value of 1, 2, 3, or 4, independently; and n is aninteger having a value of 1-5.
 16. The contact lens or intraocularimplant of claim 15, further comprising one or more therapeutic agents,prophylactic agents, diagnostic agents, or combinations thereof.
 17. Amethod for treating or preventing one or more symptoms of a disease,injury or disorder of the eye in a subject in need thereof, the methodcomprising administering to or implanting into the eye of the subjectthe dosage unit of claim 1, or the contact lens or ocular insert ofclaim
 15. 18. The method of claim 17, wherein the subject has or is atrisk of developing a disease or disorder selected from the groupconsisting of diabetes, glaucoma, corneal ectasia, dry eye, recurringcorneal erosion, Fuchs' dystrophy, keratitis, conjunctivitis, ocularherpes and Sjogren's syndrome.
 19. The method of claim 17, wherein thesubject is undergoing or has undergone a vitrectomy.
 20. The method ofclaim 17, wherein the subject has or is at risk of retinal detachment.21. The method of claim 18, wherein the self-assembling peptides orself-assembling peptidomimetics are self-assembled at the time of orafter application.
 22. The method of claim 13, wherein theself-assembling peptides or self-assembling peptidomimetics areassembled immediately prior to application.
 23. The method of claim 22,wherein the peptides are assembled by contacting the self-assemblingpeptides or self-assembling peptidomimetics with a solution of cations.24. The method of claim 17 comprising topically administering the dosageunit topically to the eye or a compartment or structure associatedtherewith.
 25. The method of claim 24 wherein the dosage is applied aseye drops, an ointment, gel, or emulsion.
 26. The method of claim 17comprising implanting or insert the device, structure, contact lens orintraocular lens into the eye or a compartment or structure associatedtherewith.
 27. The method of claim 17 comprising administering thedosage unit intravitreally.